UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

SUBJECT: EDSP Weight of Evidence Conclusions on the Tier 1 Screening Assays for the 

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 57
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 57
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: MGK 264 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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MGK 264 FINAL 

ii 

TABLE OF CONTENTS 

Abbreviations...................................................................................................iii 

Executive Summary..........................................................................................1 

I. Introduction.......................................................................................4 

II. Sources of Scientific Data and Technical Information......................................5 

A. EDSP Tier 1 Screening Assays............................................................5 

B. Other Scientifically Relevant Information (OSRI)......................................5 

III. Weight of Evidence (WoE) Evaluation.......................................................5 

A. EDSP Tier 1 Screening Assays.............................................................8 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9 

1. Effects on Estrogen Pathway.....................................................9 

2. Effects on Androgen Pathway..................................................13 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................16 

IV. Committee’s Assessment of Weight of Evidence..........................................20 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................20 

1. Tier 1 in vivo Assays.............................................................21 

2. OSRI...............................................................................21 

B. Estrogen Pathway...........................................................................23 

C. Androgen Pathway.........................................................................24 

D. Thyroid Pathway...........................................................................24 

E. Conclusions.................................................................................24 

V. EDSP Tier 2 Testing Recommendations....................................................24 

VI. References.......................................................................................26 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for MGK 264..................................................................8 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for MGK 264....................................10 

 Table 3. Androgenic/Anti-Androgenic Pathway for MGK 264.................................14 

 Table 4. Thyroid Pathway for MGK 264...........................................................18 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................29 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................41 

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iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

HPLC/MS/MS High Pressure Liquid Chromatography/Mass Spectroscopy

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

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v 

Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

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vi 

Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Executive Summary 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to 

provide the necessary empirical data to evaluate the potential of chemicals to interact with the 

estrogen (E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism 

and antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality, were considered 

in this weight of evidence (WoE) assessment. 

In determining whether MGK 264 interacts with E, A or T hormone pathways, the number and 

type of effects induced, the magnitude and pattern of responses observed across studies, taxa, 

and sexes were considered. Additionally, the conditions under which effects occur were 

considered, in particular, whether or not the dose(s) at which endocrine related responses 

occurred happened concurrently with general systemic toxicity. 

On November 19, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction 

of MGK 264 with the E, A,or T hormone pathways. The T1WoERC conclusions from the WoE 

evaluation in this report are presented by pathway (E, A and then T) beginning with the results of 

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For MGK 264, there was evidence of potential interaction with the estrogen pathway in 

mammals but not in fish. In the in vitro Tier 1 assays, MGK 264 was negative for estrogen 

receptor (ER) binding, steroidogenesis and ER transactivation assay (ERTA). MGK 264 was 

positive for inhibition of aromatase activity. In the in vivo Tier 1 assays, MGK 264 was negative 

for estrogenic activity in the uterotrophic assay. In the fish short-term reproduction assay 

(FSTRA), there were no treatment-related effects observed in females. In the female pubertal 

assay, delayed vaginal opening (VO) and delayed mean age of first estrus, and decreased 

pituitary weight was observed at a dose of 800 mg/kg/day (near the limit dose). In a post-1998 

two-generation reproduction toxicity study in rats (EDSP Tier 2 study), treatment-related effects 

seen also at a very high dose (952 mg/kg/day) included: decreased absolute and relative 

ovary/oviduct weights in the parental (P) generation; altered luteinization of the corpora lutea in 

the P and F1 offspring with an increased number of F1 females in met-/diestrus cycle; a minor but 

significant decrease in implantations with concomitant lower mean litter size at birth in F1 pups; 

and an increase in VO by 4.2 days in the absence of alterations in body weights. There were no 

estrogenic/anti-estrogenic effects noted in the other Part 158 mammalian toxicity studies. 

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For the androgen pathway, there was no convincing evidence of potential interaction of MGK 

264. It was negative in the in vitro [(androgen receptor (AR) binding and steroidogenesis)] and in 

vivo Tier 1 assays (Hershberger and male pubertal assays). In the FSTRA, while there was an 

effect on GSI, there were no other androgen-related effects observed. There were no androgenrelated effects observed in any of the Part 158 mammalian studies. 

There was evidence for potential interaction of MGK 264 with the thyroid pathway in mammals 

but not in amphibians. In the pubertal assays, histopathology showed decreased colloid area with 

a concomitant increase in follicular cell height in both males and females. However, there were 

no effects of MGK 264 noted on serum T4 or thyroid stimulating hormone (TSH) levels or on 

thyroid weights in either assays. Minor but significant decreases (p<0.05) in absolute (↓13) and 

relative (↓16%) pituitary weights were also noted at the high dose in the female pubertal assay. 

In the post 1988 two-generation reproduction study in rats, histopathology revealed increased 

incidences of thyroid gland follicular cell hypertrophy in both generations and increased 

incidences of colloidal alteration in the P, F1 males and F1 females. In a chronic toxicity/ 

carcinogenicity study in the rat, increased incidences of thyroid follicular tumors were see in 

males. There were no thyroid-related effects observed in the remaining Part 158 mammalian 

studies. In the amphibian metamorphosis assay (AMA), while there was an increase in 

normalized hind limb (HLL) on day 21, there were no treatment-related effects on HLL on day 8, 

developmental stage, or thyroid histopathology (which was relatable to a thyroid-related effect). 

The effect on day 21 limb length does not appear to be thyroid related. 

In summary, there was evidence of potential interaction of MGK 264 with the estrogen and 

thyroid pathways in mammals but not in fish or amphibians. There was no evidence of potential 

interaction with the androgen pathway either in mammals or fish. 

In the EDSP Tier 1 male and female pubertal andOSRI, there was evidence for potential 

interaction with the thyroid pathway in studies conducted with adult animals, but no such data 

exists for potential thyroid toxicity in the young animals. 

For MGK 264, the current Point of Departure (POD) of 6.1 mg/kg/day used for human health 

risk assessment is based on hepatocellular hypertrophy, the most sensitive endpoint, and 

decreased body weight. This POD was used for deriving the chronic Reference Dose (RfD) used 

for dietary risk assessment and calculating the Margins of Exposure (MOEs) used for nondietary risk assessments. This POD is approximately 50 to 160-fold lower than the doses (range, 

350 to 950 mg/kg/day) that caused estrogen- and thyroid-related effects in the pubertal assays. 

In general, since the POD and the toxicity endpoints of concern are based on data obtained from 

adult animals, there usually would be a concern that the POD may not be protective of potential 

thyroid toxicity in the young. However in the case of MGK 264, although no data exists for 

potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended 

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since: 1) the POD is approximately 50 to 160-fold lower than the doses (range, 350 to 950 

mg/kg/day) that caused histopathological lesions of the thyroid glands in the Tier 1 pubertal 

assays and OSRI; 2) the POD is based on the more sensitive endpoint (hepatotoxicity) which 

mediates the thyroid toxicity; 3) there were no effects of MGK 264 seen on serum T4 or TSH 

levels or on thyroid weights in either assays; 4) the CTA would be tested at higher doses and 

would not be expected to yield a POD lower than the level currently used for risk assessment; 

and 5) the chronic RfD and MOEs are protective of the endocrine and/or thyroid related findings 

in the Tier 1 in vivo assays and OSRI. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is not 

recommended for MGK 264 since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. As there was no 

evidence of potential interaction with the estrogen, androgen, or thyroid pathway for wildlife 

(non-mammals), ESSP Tier 2 testing is not recommended for wildlife (non-mammals). 

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I. Introduction 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to 

provide the necessary empirical data to evaluate the potential of chemicals to interact with the 

estrogen (E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism 

and antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In 

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality, were considered 

in this weight of evidence (WoE) assessment. 

In determining whether MGK 264 interacts with E, A, or T hormone pathways, the number and 

type of effects induced, the magnitude and pattern of responses observed across studies, taxa, 

and sexes were considered. Additionally, the conditions under which effects occur were 

considered, in particular, whether or not the dose(s) at which endocrine related responses 

occurred happened concurrently with general systemic toxicity. 

On November 19, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) 

of the Office of Pesticide Programs and the Office of Science Coordination and Policy conducted 

a weight-of-evidence (WoE) analysis of the potential interaction of MGK 264 with the E, A or T 

hormone pathways. The T1WoERC conclusions from the WoE evaluation in this report are 

presented by pathway (E, A and then T) beginning with the results of the Tier 1 in vitro assays 

followed by in vivo mammalian and wildlife results, then the results of the cited OSRI for 

mammalian and wildlife studies (40 CFR Part 158 and literature).

MGK 264 has a water solubility of 13.7 mg/L (at 25°C) and a octanol:water partition coefficient 

(log Kow) of 3.70. MGK 264 is stable to hydrolysis, direct aqueous photolysis, and soil 

photolysis. Aerobic and anaerobic soil metabolism are very slow (mean aerobic half-life was 341 

days). Volatilization from soil or water is not expected to be important, based on the Henry’s 

Law constant (4.44 × 10-7 atm·m3

·mol-1). MGK 264 was very mobile to moderately mobile in 

sand and sandy loam soils (Koc = 636 in sand) and essentially immobile in silt loam and clay 

loam soils (Koc = 3106 in clay loam). 

The available information considered in determining the potential interaction of MGK 264 with 

the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically 

relevant information (OSRI) such as general toxicity studies and other published articles. These 

data are summarized in Sections III.A through III.C. An analysis of the data submitted to the 

agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in 

Section IV. The EDSP Tier 2 study recommendations are presented in Section V. 

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II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the 

agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix 

1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data believed to be relevant to one or more of the Tier 1 

Screening assays were submitted as OSRI by the Test Order recipients. This included studies 

data submitted to support pesticide registration (e.g., Part 158 guideline studies). The Agency’s 

review of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on 

MGK 264 (USEPA, 2010). Since then, the Agency has also conducted a more recent search of 

available scientific literature for any additional relevant information. Summaries of the available 

OSRI are presented in Appendix 2. 

III. Weight of Evidence (WoE) Evaluation 

The principles, criteria and approach used in the WoE determination on the potential of a 

substance to interact with endocrine-related processes (i.e., E, A, or T hormone pathways) were 

as described in the WoE guidance document (USEPA, 2011) and presented at the 2013 FIFRA 

Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how 

the individual lines of evidence are assembled and integrated along two concepts (i.e., 

complementarity and redundancy) within the conceptual framework of an adverse outcome 

pathway). Broadly, there are four main steps outlined in the guidance which provide the 

foundation for WoE evaluations. The first step is to evaluate the individual studies for their 

scientific quality and relevance in evaluating potential endocrine interaction(s). The second step 

is to integrate the data along different levels of biological organization while examining the 

extent of complementarity (i.e., the concordance of endpoints within an assay that measures 

multiple endpoints) and redundancy (i.e., the concordance of endpoints/responses across assays) 

in the observed responses across these different levels of biological organization. The third step 

is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to 

evaluate whether additional testing is needed based on the evidence and conclusions described 

above. 

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the 

EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other 

scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays 

consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were 

designed specifically to evaluate a number of key biological events including potential effects on 

receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 

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effects on the HPG and HPT axes. OSRI may include published literature studies as well as 

studies conducted under USEPA (often referred to as Part 158 data) or OECD guidelines 

submitted in support of pesticide registrations. Each study is evaluated for scientific quality and 

relevance for informing interactions with the E, A, or T pathway. Additionally, the consistency 

of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple 

endpoints are measured in each assay. 

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the 

relative degree of diagnostic utility of a specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system, are considered. The collective response of the 

individual endpoints, as well as the conditions under which they were expressed, are considered 

when evaluating an overall indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate the available data 

along different levels of biological organization. Two keys elements in the integration of data, as 

well as characterizing the extent to which the available data support a hypothesis that a chemical 

has the potential to interact E, A, or T pathways, are the concepts of complementarity and 

redundancy. These two concepts provide a basis for considering the plausibility, coherence, 

strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are 

meant to evaluate whether or not a chemical can interact with E, A and T consisting of different 

levels of biological organization from a molecular initiating event, such as receptor binding, 

through potential adverse effects in apical endpoints such as sexual development and fecundity at 

the whole organism level. The extent of expression of responses at higher levels of biological 

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

After the data have been assembled and integrated, the third step is to characterize the main lines 

of evidence along with the conclusions; this characterization involves three components. The 

first component is whether the data provide relevant, robust and consistent evidence in terms of 

complementarity and redundancy, as well as biological plausibility. Second, is at what level of 

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher levels of biological organization?. Finally, under what 

conditions did the responses occur including consideration of whether the responses were 

observed in the presence of overt or systemic toxicity?. The presence of overt and/or systemic 

toxicity introduces uncertainty in the ability to distinguish effects specifically related to an 

endocrine-related effect from a non-endocrine toxic response. 

This uncertainty in distinguishing whether the responses were endocrine-related was discussed at 

the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of 

the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “In summary, the 

Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in the 

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presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in terms 

of primary interactions with the endocrine system vs. secondary effects related to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current 

approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological 

risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential 

evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as 

tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight 

decreases of ≥10% in mammals. Additionally, other effects including morphological (e.g., organ 

weights/histopathology), biochemical (e.g., blood chemistry), and other clinical signs (e.g, 

lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

extreme. In some instances, one parameter (i.e., death or >10% decrease in mammalian body 

weight) was sufficient to consider a dose/concentration to be overtly toxic. However, in other 

instances, more than one parameter was needed to determine overt toxicity. For example, in the 

FSTRA, generally, body weight decreases were considered along with other responses when 

assessing potential overt toxicity. Additionally, effects which were considered to be signs of 

systemic toxicity were also captured and these effects were generally considered as less severe 

forms of toxicity (e.g., changes in organ weights or blood chemistry). The circumstances for 

which a dose/concentration was considered overtly toxic for a particular study are described in 

Section IV.A. 

In summary, EPA considers multiple lines of evidence in including the observed responses in 

the Tier 1 assays and OSRI in the context of a chemical’s physical/chemical properties and its 

known modes of action in its overall characterization of a chemical’s potential to interact with 

the E, A or T pathway. Adequately addressing the three main components described above is 

fundamental to the WoE process and in determining whether additional data are needed. In 

addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their 

potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or 

conflicting data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, 

additional data are needed and why. Assessing the need for additional data is based on a caseby-case analysis which took all available toxicity data into account. 

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The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 

assays and OSRI which are depicted in Tables 2 - 4. These tables contain data that are 

considered scientifically and biologically relevant with regard to a treatment-related effect 

which supports a conclusion of whether a substance has the potential to interact with the E, A, 

or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for 

each respective pathway (E, A or T) but are not reported in the table for E, A or T. 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the 

agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix 

1. 

 Table 1. Tier 1 Screening Assays for MGK 264 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirment Satisfied 

(MRID 48618003) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OSCPP 890.1300; 

OECD 455 

Requirment Satisfied 

(MRID 48618004) 

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirment Satisfied 

(MRID 48618001) 

Steroidogenesis Assay (Human cell line 

H295R) 

OSCPP 890.1550; 

OECD 456 

Requirment Satisfied 

(MRID 48672301) 

Aromatase Assay (human recombinant 

microsomes) OSCPP 890.1200 Requirment Satisfied (MRID 48618002) 

Uterotrophic Assay (Rat) OSCPP 890.1600; 

OECD 440 

Requirment Satisfied 

 (MRID 48618006) 

Hershberger Assay (Rat) OSCPP 890.1400; 

OECD 441 

Requirment Satisfied 

(MRID 48618005) 

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirment Satisfied 

(MRID 48686301) 

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirment Satisfied 

(MRID 48686301) 

Fish Short-term Reproduction Assay OSCPP 890.1350; 

OECD 229 

Requirment Satisfied 

(MRID 48923701) 

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; 

OECD 231 

Requirment Satisfied 

(MRID 48701701) 

Page 16 of 57
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Page 9 of 49 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis 

1. Effects on Estrogen Pathway 

The potential for MGK 264 to interact with the estrogen pathway is summarized in Table 2. The 

various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects. This 

table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in 

the presence of overt toxicity are discussed in the text but are not reported in the table and not 

considered further in the WOE assessment. 

The Tier 1 in vitro ER binding, ERTA and steroidogenesis and in vivo uterotrophic assays were 

negative. MGK 264 was positive for aromatase inhibition with an estimated IC50 of 10−4.7 M. 

In the FSTRA, while it is noted that female fish (including controls) had findings of oocyte 

atresia (5-14%) sometimes associated with inflammation and presence of protozoal spores, 

fecundity was adequate in the assay, and there were no treatment-related effects observed 

(including fecundity) for the females up to 0.107 mg a.i./L, the highest concentration tested. 

In the female pubertal rat assay at 800 mg/kg/day, initial and complete VO were delayed 

(p<0.001) by 3.1 and 3.4 days, respectively, and mean age of first estrus was delayed (p<0.05) 

by 2.5 days. Additionally, unadjusted and adjusted body weights at VO were increased (p<0.01) 

by 16-17% suggesting that the delay in VO was not a growth effect. In addition, a 13% decrease 

in ovarian and pituitary weight was seen at the high dose. Increased absolute, adjusted and 

relative liver weights was observed at both 400 and/or 800 mg/kg/day. 

In the 2009 Part 158 two-generation reproduction toxicity study in rats, effects observed in 

females at the high dose (952 mg/kg/day) included decreased absolute and relative ovary/oviduct 

weights (16-20%) in the P generation, altered luteinization of the corpora lutea in the P (8/25) 

and F1 (11/25) females compared to 0/25 controls, with an increased number of F1 females in 

met-/diestrus cycle at the high dose (16/25 treated vs. 8/25 controls). Also, there was a small but 

significant decrease in implantations with concomitant lower mean litter size at birth in F1 pups. 

At the high dose, the age at VO was increased by 4.2 days with body weights at VO unaffected 

by treatment. Additionally, increased absolute liver weights with corroborating gross and 

microscopic liver findings, increased kidney weights with increased storage of brownish pigment 

in the cortical tubules were observed at the mid and/or high dose in this study. 

There were no estrogenic/anti-estrogenic effects noted in the other Part 158 mammalian toxicity 

studies.

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Page 10 of 49 

Table 2: Estrogenic/Anti-Estrogenic Pathway for MGK 264 Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for MGK 2641Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

EDSP Tier 1 Assay 

ER Binding (MRID 48618003) N ERTA (MRID 48618004) N4 Aromatase (MRID 48618002) P Steroidogenesis (MRID 48672301) N Uterotrophic (MRID 48618006) N Female Pubertal Rat (MRID 48686301) N ↓13% (H) N ↓13 % (H) N ↑3.4 d wt: ↑17% (H)6 FSTRA (MRID 48923701) NE N N N N N 

OSRI 

Two-Generation Reproduction Toxicity (Rat; MRID 48084901) 

 N 

↓16-

20% 

(H) 

P7 

 (H) N P8 (H) ↑4.2 d ↑wt (H) P9 (H) 

Two-Generation Reproduction Toxicity (Rat; MRID 42155701) 

 NE NE N NE N N 

Developmental Toxicity (Rat; MRID 41660302) 

 N 
MGK 264 FINAL 

Page 11 of 49 

Table 2: Estrogenic/Anti-Estrogenic Pathway for MGK 264 Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for MGK 2641Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

Developmental Toxicity (Rabbit; MRID 40352301) 

N 

Chronic Toxicity/ Carcinogenicity (Rat; MRID 43005301) 

 NE N N NE 

Carcinogenicity (Mouse; MRID 42093802) 

NE N N NE 

Chronic Toxicity (Dog; MRID 42148102) NE N N N Subchronic Oral Toxicity (Rat; MRID 42970601) 

 NE N NE NE 

Subchronic Inhalation Toxicity (Rat; MRID 43309001) 

NE N NE NE 

Subchronic Dermal Toxicity (Rabbit; MRID 42742201) 

NE NE N NE 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=Hig

↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in 

weight are absolute unless otherwise indicated. 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the O

in this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by an 

details see Section IV. A 
MGK 264 FINAL 

Page 12 of 49 

4. Although the responsiveness of the cells to the very weak positive control 17α-methyltestosterone was lower than the expected va

were similar to those of the negative control corticosterone (0.7-3.9% of positive control (PC)) and not comparable to the respons

methyltestosterone, which was able to reach a maximum of 8.1-52.8% PC. MGK 264 was only able to reach a maximum of 1.2-5the highest concentration possible based on cytotoxicity. The reference chemicals 17β-estradiol and 17α-estradiol (weak estrogen

in both runs. Because the RPCMax < PC10 in both assay runs, MGK 264 was considered negative in this assay. 

5. One animal from the 1000 mg/kg/day group was euthanized moribund on Day 3, and terminal body weights were decreased (p<0

mg/kg/day. In addition, animals in the 1000 mg/kg/day group exhibited adverse clinical signs of toxicity (uncoordinated movemehunched posture, twitching, piloerection, ungroomed appearance, a red nasal discharge, and/or diarrhea) following dosing. 

6. At 800 mg/kg/day, ages at initial and complete VO were delayed (p<0.001) by 3.1 and 3.4 days, respectively, and mean age of fir

7. In the female genital tract, altered luteinization of the corpora lutea was observed at 6400 ppm in the P (8/25) and F1 (11/25) anim

controls. 

8. There were an increased number of F1 females in met-/diestrus cycle at 6400 ppm (16/25 treated vs. 8/25 controls). 

9. At 6400 ppm, there was a small but significant decrease in implantations with concomitant lower mean litter size at birth in F1 pup

litter size is within the historical control range, a relationship to treatment cannot be ruled out due to the histopathological finding

the parental females. 

P Positive findings N Negative findings (in vitro)/No effect (in vivo) 

NE Not examined 
MGK 264 FINAL 

Page 13 of 49 

2. Effects on Androgen Pathway 

The potential for MGK 264 to interact with the androgen signaling pathway is summarized in 

Table 3. The various targets of the androgen pathway across the relevant Tier 1 assays are 

delineated so as to facilitate determination of potential for androgenic, anti-androgenic or HPG 

axis effects. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects 

that occurred in the presence of overt toxicity are discussed in the text but are not reported in the 

table and not considered further in the WOE assessment. 

In the in vitro Tier 1 assays, MGK 264 was negative in both the AR binding and steroidogenesis 

assays. In the in vivo Tier 1 assays, MGK 264 was negative in the Hershberger for both 

androgenic and anti-androgenic activity, and there were no treatment-related effects on 

androgen-related endpoints in the male rat pubertal assay. In the FSTRA, male GSI was 

increased (p<0.05) by 45% at the highest concentration tested (0.107 mg a.i./L). It is noted that 

gonadal histopathology findings (e.g, increases in testicular degeneration) were reported in all 

treatments (including controls); however, they did not appear to be treatment-related and all 

other endpoints, except GSI, were unaffected by treatment. 

In the 2009 Part 158 two-generation reproduction toxicity study in rats (MRID 48084901), 

absolute and relative seminal vesicle weights were increased (p<0.05) by 9-10% at the highest 

dose tested (650 mg/kg/day); however, the effect occurred at a dose that resulted in overt toxicity 

(>10% decrease in body weight). No other androgenic effects were noted in the study. There 

were no androgenic or anti-androgenic effects observed in any of the remaining Part 158 

mammalian studies. 

 

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Table 3: Androgenic/Anti-Androgenic Pathway for MGK 264 Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for MGK 2641Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin 

EDSP Tier 1 Assay 

AR Binding (MRID 48618001) N Steroidogenesis (MRID 48672301) N Hershberger (MRID 48618005) NE N Male Pubertal Rat (MRID 48686301) N N N N N N N N FSTRA (MRID 48923701) NE 

↑45% 

GSI 

(H) 

N N N N 

OSRI 

Two-Generation Reproduction Toxicity (Rat; MRID 48084901) 

N N N N N 

SV 

↑9-10% 

(H) 

 N 

Two-Generation Reproduction Toxicity (Rat; MRID 42155701) 

NE N NE N NE NE 

Chronic Toxicity/ Carcinogenicity (Rat; MRID 43005301) 

N N NE N N NE 
MGK 264 FINAL 

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Table 3: Androgenic/Anti-Androgenic Pathway for MGK 264 Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for MGK 2641Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Sex Steroid 

Hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin 

Carcinogenicity (Mouse; MRID 42093802) 

N N NE N NE N 

Chronic Toxicity (Dog; MRID 42148102) 

N N NE NE NE NE 

Subchronic Oral Toxicity (Rat; MRID 42970601) 

N NE NE NE NE NE 

Subchronic Inhalation Toxicity (Rat; MRID 43309001) 

N N N N NE NE 

Subchronic Dermal Toxicity (Rabbit; MRID 42742201) 

N N NE N NE NE 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High 

or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prost(DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the O

in this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by an 

details see Section IV. A 

4. At 350 and 700 mg/kg/day, increased (p<0.05) absolute, adjusted, and relative liver weights (↑20-23% and ↑39%, respectively). A

mg/kg/day, increases (p<0.05) were noted in relative kidney weights (↑8%) and serum BUN, SDH, and total protein. 

P Positive findings N Negative findings (in vitro)/No effect (in vivo); 

NE Not examined
MGK 264 FINAL 

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C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis 

The current EDSP Tier 1 battery does not have a specific in vitro assay to detect chemicals with 

the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it 

does include three in vivo assays that provide data to detect changes in the HPT axis, i.e., the 

pubertal male and female (rat) assays, and the AMA (frog). 

The potential for MGK 264 to interact with the thyroid signaling pathway is summarized in 

Table 4. The various targets of the thyroid pathway across the relevant Tier 1 assays are 

delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table 

also includes HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the 

presence of overt toxicity are discussed in the text but are not reported in the table and not 

considered further in the WOE assessment. 

In the EDSP Tier 1 pubertal assays, decreased colloid area with a concomitant increase in 

follicular cell height was observed microscopically at both dose levels in both the male (350 and 

700 mg/kg/day) and female (400 and 800 mg/kg/day) pubertal assays. However, there were no 

effects of MGK 264 noted on serum T4 or TSH levels or on thyroid weights in either assays, and 

systemic toxicity, presenting primarily as increased liver weights, was noted at both doses in 

both assays. Small decreases (p<0.05) in absolute (↓13) and relative (↓16%) pituitary weights 

were also noted at the high dose in the female pubertal assay. 

In the AMA, nearly all exposed individuals were developed beyond NF stage 60 by Day 21 

(referred to as ‘late-stage’). The only effect observed was an increase (p<0.05) of 28% in 

normalized hind limb length (HLL) at Day 21 at the highest concentration (0.106 mg a.i./L). All 

other parameters were unaffected by treatment. Animals were stage matched to the greatest 

extent possible for thyroid histopathological evaluation. Histopathological findings included 

mild follicular cell hypertrophy and hyperplasia in the negative control and all treatment groups. 

Specifically, for follicular cell hypertrophy, the number of mild diagnoses in the control group 

(7) was higher than the number for the mid and high treatment groups (6 and 5, respectively). 

Similarly for follicular cell hyperplasia diagnoses, there were more diagnoses of mild severity in 

the control group (5) than in the treatment groups (2, 1, and 0, respectively). Although a 

decrease in the incidence of follicular cell hypertrophy or hyperplasia was observed in the 

treatment groups relative to the control, given that there were no effects on developmental stage 

or effect on hind limb length on Day 7 (which may be more indicative of a potential interaction 

with the thyroid than on Day 21), the effects observed do not appear to be thyroid-related. 

In a 2009 Part 158 two-generation reproduction study in rats, increased incidences of thyroid 

gland follicular cell hypertrophy were observed in both generations in the mid dose males and 

high dose females (159 and 952 mg/kg/day, respectively). Increased incidences of colloidal 

alteration were noted in the P males at the high dose (650 mg/kg/day), in the F1 males at the mid 

and high dose, and in the F1 females at the high dose. Overt and systemic toxicity, presenting 

Page 24 of 57
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Page 17 of 49 

primarily as decreased body weights with increased liver weights, were also observed at these 

dose levels. In a chronic toxicity/carcinogenicity study in the rat, an increase in thyroid follicular 

tumors in males were observed in males. The incidence for adenomas alone were 6.7%, 8.3% 

and 10% at 50, 150, and 450 mg/kg/day, respectively, compared to 1.7-3.3% in the controls 

(n=60/group). The incidence for combined adenomas and carcinomas were 10%, 11.7% and 15% 

at 50, 150, and 450 mg/kg/day, respectively, compared to 1.7-6.7% for the controls 

(n=60/group). Again, overt and systemic toxicity, presenting primarily as decreased body 

weights were noted at 450 mg/kg/day and increased liver weights were observed at 50, 150, and 

450 mg/kg/day. There were no thyroid-related effects observed in the remaining Part 158 

mammalian studies. 

 

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Table 4. Thyroid Pathway for MGK 264

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for MGK 2641

Study Type/ 

Literature Citation 

Thyroid receptors Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, SVL) Systemic Toxicity Observed2 

Overt Toxicity Observed3

EDSP Tier 1 Assays 

Male Pubertal Rat 

(MRID 48686301) N P4(L, H) N N N 

X5

 

liver: 

↑23, 39% 

(L, H) 

N 

Female Pubertal 

Rat (MRID 

48686301) 

 N P6

(L, H) N N ↓13% (H) 

P7

 

liver: 

↑26, 44% 

(L, H) 

N 

AMA 

(MRID 48701701) N 

HLL 

↑28%

(H)8 

N N 

OSRI

Two-Generation 

Reproduction 

Toxicity (Rat; 

MRID 48084901) 

 N P9

(M, H) N X 

(L, M, H) N 

Chronic Toxicity/ 

Carcinogenicity 

(Rat; MRID 

43005301) 

 NE P10 

(M, H♂) NE X 

(L, M, H) 

X 

(♀ H) 

Carcinogenicity 

(Mouse; MRID 

42093802) 

 NE N NE X 

(M, H) N 

Chronic Toxicity 

(Dog; MRID 

42148102) 

 N N N X 

(H) N 

Subchronic 

Inhalation Toxicity 

(Rat; MRID 

43309001) 

 NE N NE X 

(ML, MH, H) N 

Subchronic Dermal 

Toxicity (Rabbit; 

MRID 42742201) 

 NE N NE N N 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high 

treatment, H=High treatment, Medium treatment (M), High treatment (H). Arrows (↓ or ↑) indicate the 

direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not 

applicable in this assay. 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The 

systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI 

is indicated by an X in this column. For details see Section IV. 

Page 26 of 57
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4. Mean colloid area was decreased (p<0.05) at 350 mg/kg/day; at 700 mg/kg/day, the incidence/grade of mean 

colloid area at 700 mg/kg/day was similar (NS) to that observed at 350 mg/kg/day. Follicular cell height was 

slightly increased (NS) at 350 and 700 mg/kg/day. There was an increased incidence of minimal to moderate 

eosinophilic colloid in the thyroids of the 350 and 700 mg/kg/day males. 

5. At 350 and 700 mg/kg/day, increased (p<0.05) absolute, adjusted, and relative liver weights (↑20-23% and 

↑39%, respectively). Additionally at 700 mg/kg/day, increases (p<0.05) were noted in relative kidney weights 

(↑8%) and serum BUN, SDH, and total protein. 

6. Mean colloid area was decreased (p<0.05) and mean follicular height was increased (p<0.05) in the 400 and 

800 mg/kg/day groups, with an increased incidence of minimal to moderate eosinophilic colloid. 

7. Absolute, adjusted, and relative liver weights were increased (p<0.001) by 21-26% and 40-44% at 400 and 800 

mg/kg/day, respectively. 

8. The increase (Jonckheere-Terpstra, p<0.05) in normalized HLL (HLL) based on analysis using late stage only 

individuals (NF stage ≥ 60) as nearly all animals were ‘late-stage’ by Day 21. 

9. In the thyroid glands, increased incidences of follicular cell hypertrophy were observed in both generations in 

the 1600 ppm males and 6400 ppm females. Increased incidences of colloidal alteration were noted in the P 

males at 6400 ppm, in the F1 males at 1600 and 6400 ppm, and in the F1 females at 6400 ppm. 

10. Increased thyroid follicular tumors were seen in males with a response of 2 (3.3%), 1 (1.7%), 4 (6.7%), 5 

(8.3%) and 6 (10%) for adenomas alone, and 4 (6.7%), 1 (1.7%), 6 (10%), 7 (11.7%) and 9 (15%) for 

combined adenomas and carcinomas for the two control, 50, 150 and 450 mg/kg/day dose groups respectively 

(n=60/dose group). 

P Positive findings 

N Negative findings 

NE Not examined 

 

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IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight of evidence (WoE) determination on the 

potential of MGK 364 to interact with endocrine related processes (i.e., E, A, or T hormonal 

pathways) as well as recommendations regarding Tier 2 testing.. The results of the Tier 1 assays 

are considered, along with other scientifically relevant information (e.g., 40 CFR Part 158 test 

guidelines and published or publicly available peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and 

OSRI), then performing an integrated analysis of those lines of evidence. 

The WoE evaluation includes considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, coherence, and interrelationships 

among the measured endpoints within and across studies. The available findings from standard 

toxicology studies on the substance may contribute to the WoE evaluation in helping elucidate if 

effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or 

alternatively sequelae of systemic effects. Endocrine modes of action may elicit a number of 

phenotypic consequences other than those evaluated in the Tier 1 assays. 

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to 

whether or not the responses are related through an endocrine pathway, therefore non-endocrine 

toxic responses (including but not limited to mortality or body weight changes) are also 

considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated 

with weight of evidence evaluation of the results of the Tire 1 assays stated that “In summary, 

the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in 

the presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in 

terms of primary interactions with the endocrine system vs. secondary effects related to toxicity 

in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI 

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and 

OSRI studies are described below. Generally, one parameter (i.e., death or >10% decrease in 

mammalian body weight) is sufficient for a dose/concentration to be considered overtly toxic. 

However, in other instances, more than one parameter was needed to determine overt toxicity. 

Effects which were considered to be signs of systemic toxicity were generally less severe forms 

of toxicity (e.g., changes in organ weights or blood chemistry).

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1. Tier 1 in vivo Assays 

Toxicity was not observed in the AMA at concentrations up to 0.106 mg a.i./L, in the FSTRA at 

concentrations up to 0.107 mg a.i./L, or in the Hershberger assay at dose levels up to 850 

mg/kg/day. 

In the pubertal rat assays, systemic toxicity, presenting primarily as increased liver weights, was 

noted in both the male (350 and 700 mg/kg/day) and female (400 and 800 mg/kg/day) pubertal 

assays. 

In the uterotrophic assay, animals in the 1000 mg/kg/day group exhibited adverse clinical signs 

of toxicity (uncoordinated movement, decreased movement, hunched posture, twitching, 

piloerection, ungroomed appearance, a red nasal discharge, and/or diarrhea) following dosing. 

Additionally, one animal from the 1000 mg/kg/day group was euthanized moribund on Day 3. 

Body weights were decreased (p<0.05) by 10% on Day 4 in the 1000 mg/kg/day group compared 

to the vehicle controls, resulting in a mean overall body weight loss (p<0.05) of −8.9 g, 

compared to the vehicle control group (16.5 g gain). 

2. OSRI 

In a two-generation rat reproduction toxicity study, body weight decreases (p<0.05) during premating were noted at 1250 (↓5-10%), 2500 (↓5-7%), and 10,000 (↓9-15%) ppm in the F1 dams 

and at 2500 (↓↓6-8%) and 10,000 (↓6-10%) ppm in the P dams (equivalent to 61/73, 125/151, 

and 649/759 mg/kg/day in males/females). In the F1 males, body weighs were decreased 

(occasionally p<0.05) at 2500 (↓4-8%) and 10,000 (↓9-20%) ppm. Hepatocellular hypertrophy 

was evident at all doses in the F1 parental group and at higher levels in the P parental group. 

Decreased body weight during lactation was evident at all doses with there being decreases of 

about 5% to 14% (all p < 0.05) at 1,250 ppm, 5% to 13% at 2,500 ppm and 26% to 31% at 

10,000 ppm. Following lactation, the pups gained weight comparable to the controls. 

In a post-1998 two-generation rat reproduction toxicity study (EDSP Tier 2 study), systemic and 

overt toxicity were observed at 1600 and 6400 ppm (equivalent to 159/225, and 650/952 

mg/kg/day in males/females). The 6400 ppm F1 group body weights and body weight gains were 

decreased (p<0.05) and relative (to body weight) food consumption was increased (p<0.05) 

during pre-mating. Body weights were decreased (p<0.05) in the 1600 ppm F1 females at the end 

of the second gestation, and at 6400 ppm at the end of both gestations and on post-natal day 

(PND) 4. Relative food consumption was dose-dependently increased in the 1600 and 6400 ppm 

F1 dams during the 2nd mating. Absolute and relative liver weights were dose-dependently 

increased, with corroborating gross and microscopic liver finding in both sexes and/or 

generations. Microscopic findings were noted in the kidney at 1600 ppm and above in both sexes 

and/or generations. In the F2a litters, small pups relative to other pups in the litter were observed 

with a higher frequency at 6400 ppm. Additionally at 6400 ppm, decreased (p≤0.05) offspring 

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body weights were observed in both sexes in the F1 and F2b litters and in the F2a females on PND 

21 and 28 (↓11-17%), and body weights were decreased by 14% (p<0.05) on PND 14 in the F2b

females. 

In a rat developmental toxicity study, increased salivation was noted at 1000 mg/kg/day as a predose observation in four dams and post-dosing in 23 dams. Body weight gains were decreased by 

25% (p<0.01) during GD 6-15 at 1000 mg/kg/day. The pregnancy rate was decreased at 1000 

mg/kg/day (68% treated vs. 80% controls); however, there was no corroborating evidence of 

effects on fertility. 

In a rabbit developmental toxicity study, an increase in the incidence of excessive salivation was 

seen at 100 mg/kg/day (44% of the does vs. 0% in the controls); this occurred on consecutive 

days in the middle of the dosing period and is considered a response to treatment. 

In a rat chronic toxicity/carcinogenicity study, final body weights were decreased 8-9% in the 

males and 18-21% in the females at the high dose (450 mg/kg/day). Decreases in body weight 

gains were also observed at this dose, reaching 58% in males and 50% in females during Weeks 

52-78. Food consumption was also generally decreased in the 450 mg/kg/day females. Absolute 

liver weights were increased (p<0.05) by 14-45% in the males at ≥50 mg/kg/day and by 9-22% 

in females at ≥150 mg/kg/day. At 150 mg/kg/day and above, pathological changes were 

observed in the liver (hepatocyte hypertrophy in both sexes and portal bile duct proliferation and 

bile stasis, spongiosis hepatitis and cysts in females) and kidney (brown pigment in females). At 

450 mg/kg/day, additional pathology was noted in the liver (bile duct proliferation, cysts and 

altered eosinophilic foci in males and spongiosis hepatitis in males) and kidney (brown pigment 

and cysts in males); and kidney, heart, and brain weights were increased. 

In a mouse carcinogenicity study, at 400 mg/kg/day and above, absolute and relative (to body) 

liver weights were dose-dependently increased (p<0.01) in the males by 13-37% and 22-46%, 

respectively, compared to the controls. In the females, only relative (to body) liver weight at 

these doses was significantly (p<0.01) increased (↑11-16%), although absolute liver weights 

were slightly elevated at 400 (↑8%) and 800 mg/kg/day (↑6%). At 400 mg/kg/day there was 

increased calculi in the gall bladder and intrahepatic bile stasis in males. At 800 mg/kg/day there 

was an increase in hepatocellular hypertrophy, calculus in bile duct in males and females and 

portal bile duct proliferation, portal mononuclear cell infiltration, spongiosis hepatis, cysts and 

vacuolar change in males and hyperplastic nodules in males and females. Increases in liver 

adenomas were also observed in the 400 and 800 mg/kg/day males. Females at 400 and 800 

mg/kg/day also had adenomas and a carcinoma which did not reach statistical significance, but 

these liver tumors are rare in female CD-1 mice. 

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In a one-year dog chronic toxicity study, the only effects noted were brown pigment deposition 

in the liver of both sexes (3/4 males and females vs. 0/4 in controls) and hepatocellular 

hypertrophy in 2/4 males seen at 1000 ppm dose level (equivalent to 34.9/32.5 mg/kg/day in 

males/females). 

In a rat subchronic toxicity study, at 1000 mg/kg/day, body weights were decreased (p<0.05) by 

14% in the males and 17% in the females at Week 13. Body weight gains were also decreased at 

500 and 1000 mg/kg/day in both sexes compared to controls (10% and 21% in males and 

18% and 33% in females). Food consumption was frequently decreased (p<0.05) at 500 and 

1000 mg/kg/day in males and females throughout the study. Mean total protein values and 

globulin levels were increased at 250 mg/kg/day and above, and cholesterol levels were 

increased at 500 mg/kg/day and above in the males; mean total protein values, globulin levels, 

and cholesterol levels were increased at ≥500 mg/kg/day in the females. Absolute and relative (to 

body) liver weights were increased in both sexes at ≥250 mg/kg/day. At necropsy, enlarged liver 

was noted in males at 500 and 1000 mg/kg/day. 

In a rat subchronic inhalation toxicity in rats, clinical signs of toxicity observed were red facial 

stains and decreased activity. Metaplasia/hyperplasia/hypertrophy of the mucosal epithelium 

were observed in the larynx, ventral diverticulum, and nasoturbinates, and in the goblet cells in 

the respiratory epithelium and nasopharynx. 

No systemic toxicity was observed in a rabbit subchronic dermal toxicity study. 

B. Estrogen Pathway 

There was evidence of potential interaction of MGK 264 with the estrogen pathway in mammals 

but not in fish. In the in vitro Tier 1 assays, MGK 264 was negative for ER binding, 

steroidogenesis and ERTA. MGK 264 was positive for inhibition of aromatase activity. In the in 

vivo Tier 1 assays, MGK 264 was negative for estrogenic activity in the uterotrophic assay. In 

the FSTRA, there were no treatment-related effects observed in females. In the female pubertal 

assay, delayed VO and delayed mean age of first estrus, and decreased pituitary weight was 

observed at a dose of 800 mg/kg/day (near the limit dose). 

In a two-generation reproduction toxicity study in rats conducted under the 1998 revised test 

guidelines, treatment-related effects seen also at a very high dose (952 mg/kg/day) included: 

decreased absolute and relative ovary/oviduct weights in the P generation; altered luteinization of 

the corpora lutea in the P and F1 offspring with an increased number of F1 females in met-

/diestrus cycle; a minor but significant decrease in implantations with concomitant lower mean 

litter size at birth in F1 pups; and an increase in VO by 4.2 days in the absence of alterations in 

body weights. There were no estrogenic/anti-estrogenic effects noted in the other Part 158 

mammalian toxicity studies. 

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C. Androgen Pathway 

There was no convincing evidence of potential interaction with the androgen pathway. MGK 264 

was negative in the in vitro (AR binding and steroidogenesis) and in vivo Tier 1 assays 

(Hershberger and male pubertal assays). In the FSTRA, while there was an effect on GSI, there 

were no other androgen-related effects observed. There were no androgen-related effects 

observed in any of the Part 158 mammalian studies. 

D. Thyroid Pathway 

There was evidence for potential interaction of MGK 264 with the thyroid pathway in mammals 

but not in amphibians. In the AMA, while there was an increase in normalized hind limb (HLL) 

on day 21, there were no treatment-related effects on HLL on day 8, developmental stage, or 

thyroid histopathology (which was relatable to a thyroid-related effect).The effect on day 21 limb 

length does not appear to be thyroid related. 

In the pubertal assays, histopathology showed decreased colloid area with a concomitant increase 

in follicular cell height in both males and females. However, there were no effects of MGK 264 

noted on serum T4 or TSH levels or on thyroid weights in either assays. Minor but significant 

decreases (p<0.05) in absolute (↓13) and relative (↓16%) pituitary weights were also noted at the 

high dose in the female pubertal assay. In the post 1988 two-generation reproduction study in 

rats, histopathology revealed increased incidences of thyroid gland follicular cell hypertrophy in 

both generations and increased incidences of colloidal alteration in the P, F1 males and F1

females. In a chronic toxicity/ carcinogenicity study in the rat, increased incidences of thyroid 

follicular tumors were see in males. There were no thyroid related effects observed in the 

remaining Part 158 mammalian studies. 

E. Conclusions 

There was evidence of potential interaction of MGK 264 with the estrogen and thyroid pathways 

in mammals but not in fish or amphibians. There was no evidence of potential interaction with 

the androgen pathway either in mammals or wildlife (non-mammals). 

V. EDSP Tier 2 Testing Recommendations 

In the EDSP Tier 1 male and female pubertal andOSRI, there was evidence for potential 

interaction with the thyroid pathway in studies conducted with adult animals, but no such data 

exists for potential thyroid toxicity in the young animals. 

For MGK 264, the current Point of Departure (POD) of 6.1 mg/kg/day used for human health 

risk assessment is based on hepatocellular hypertrophy, the most sensitive endpoint, and 

decreased body weight. This POD was used for deriving the chronic Reference Dose (RfD) used 

for dietary risk assessment and calculating the Margins of Exposure (MOEs) used for nonPage 32 of 57
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dietary risk assessments. This POD is approximately 50 to 160-fold lower than the doses (range, 

350 to 950 mg/kg/day) that caused estrogen- and thyroid-related effects in the pubertal assays. 

In general, since the POD and the toxicity endpoints of concern are based on data obtained from 

adult animals, there usually would be a concern that the POD may not be protective of potential 

thyroid toxicity in the young. However in the case of MGK 264, although no data exists for 

potential thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended 

since: 1) the POD is approximately 50 to 160-fold lower than the doses (range, 350 to 950 

mg/kg/day) that caused histopathological lesions of the thyroid glands in the Tier 1 pubertal 

assays and OSRI; 2) the POD is based on the more sensitive endpoint (hepatotoxicity) which 

mediates the thyroid toxicity; 3) there were no effects of MGK 264 seen on serum T4 or TSH 

levels or on thyroid weights in either assays; 4) the CTA would be tested at higher doses and 

would not be expected to yield a POD lower than the level currently used for risk assessment; 

and 5) the chronic RfD and MOEs are protective of the endocrine and/or thyroid related findings 

in the Tier 1 in vivo assays and OSRI. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is not 

recommended for MGK 264 since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. As there was no 

evidence of potential interaction with the estrogen, androgen, or thyroid pathway for wildlife 

(non-mammals), ESSP Tier 2 testing is not recommended for wildlife (non-mammals). 

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VI. References

Blair, M. (1989) 13 week dietary range-finding toxicity study in rats with MGK 264. 

International Research and Development Co., Mattawan, MI. Laboratory Project Id. 551-028, 

August 9, 1989. Unpublished. (MRID 42970601) 

Blair, M. (1991) One year dietary toxicity study in dogs with MGK 264. International Research 

and Development Co., Mattawan MI. Laboratory Study No. 551-031, December 16, 1991. 

Unpublished. (MRID 42148102) 

Blair, M. (1991) Eighteen month dietary oncogenicity study in mice with MGK 264 insecticide 

synergist. International Research and Development Co., Mattawan, MI. Laboratory Study No.: 

551-011, June 13, 1991. Unpublished. (MRID 42093802) 

Eiben, R. (2009) MGK 264: two-generation reproduction study in Wistar rats by administration 

in the diet. Bayer Shering Pharma AG, Wuppertal, Germany. Laboratory Report/Study No. 

AT05546/T1073335, September 30, 2009. Unpublished. (MRID 48084901) 

Fort, D. (2012) MGK 264: 21-Day amphibian metamorphosis assay. Fort Environmental 

Laboratories, Inc., Stillwater, OK. Sponsored by McLaughlin Gormley King Co., Minneapolis, 

MN. Laboratory Report No. MGK01-00273, July 24, 2012. Unpublished. (MRID 48701701) 

Fort, D. (2012) MGK 264: 21-Day fish reproduction assay. Fort Environmental Laboratories, 

Inc., Stillwater, OK; Golden Pacific Laboratories, Inc., Fresno, CA; Experimental Pathology 

Laboratories, Inc., Sterling, VA. Sponsored by McLaughlin Gormley King Company, 

Minneapolis, MN. Laboratory Report No. MGK01-00274, August 30, 2012. Unpublished. 

(MRID 48923701) 

Goldenthal, E. (1993) 24-Month dietary chronic toxicity and oncogenicity study in the rat with 

MGK 264. International Research and Development Co., Mattawan, MI. Laboratory Project No. 

551-030, October 8, 1993. Unpublished. (MRID 43005301) 

Lancaster, S., and Husband, R. (1993) MGK 264 synergist 13-week dermal toxicity study in the 

rabbit. Toxicol Laboratories Ltd., England. Laboratory Study No. MCA/17/92, March 19, 1993. 

Unpublished. (MRID 42742201) 

Newton, P. (1993) A subchronic (3-month) inhalation toxicity study of MGKR 264 insecticide 

synergist in the rat via whole body exposures. Unpublished study conducted by Pharmaco:LSR, 

East Millstone, NJ; Sponsored by McLaughlin Gormley and King Company. Laboratory Study 

No.: 91-8364. June 24, 1994. (MRID 43309001) 

Schardein, J. (1987) Teratological study of MGK 264 insecticide synergist administered orally to 

albino rabbits. International Research and Development Corporation, Mattawan, MI. Sponsored 

Page 34 of 57
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Page 27 of 49 

by McLaughlin Gormley King Company, Minneapolis, MN. Laboratory Project Id. 551-015, 

August 31, 1987 Unpublished. (MRID 40352301) 

Schardein, J. (1990) Developmental toxicity study in rats. International Research and 

Development Corporation, Mattawan, MI. Sponsored by McLaughlin Gormley King Company, 

Minneapolis, MN. Laboratory Identification No. 551-026, June 8, 1990. Unpublished. (MRID 

41660302) 

Schardein, J. (1991) Two-generation reproduction study of MGK 264 in the albino rat. 

International Research and Development Corporation, Mattawan, MI. Laboratory Study No. 551-

027, December 18, 1991. Unpublished. (MRID 42155701) 

USEPA (2010) MGK 264 - Report of the Endocrine Disruptor Review Team - Test Order#: 

EDSP- 057001- 250. Office of Chemical Safety and Pollution Prevention Science Coordination 

and Policy. U.S. Environmental Protection Agency, Washington, DC. 

USEPA (2011) Endocrine Disruptor Screening Program – Weight of Evidence: evaluating 

results of EDSP Tier 1 screening to identify the need for Tier 2 testing. Office of Chemical 

Safety and Pollution Prevention Science Coordination and Policy. U.S. Environmental Protection 

Agency, Washington, DC. 

USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting 

held July 30 - August 1, 2013 on Weight-of-Evidence: Evaluating Results of EDSP Tier 1 

Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection 

Agency, Washington, DC. 

Wagner, H. (2012) MGK 264: H295R steroidogenesis assay. CeeTox, Inc., Kalamazoo, MI. 

Laboratory Study No. 9146V-100355STER, January 26, 2012. Unpublished. (MRID 48672301) 

Wilga, P. (2012) MGK 264: human recombinant aromatase assay. CeeTox, Inc., Kalamazoo, MI. 

Laboratory Report No. 9146V-100355AROM, January 11, 2012. Unpublished. (MRID 48618002) 

Willoughby, J. (2012) MGK 264: androgen receptor binding (rat prostate cytosol). CeeTox, Inc., 

Kalamazoo, MI. Laboratory Project Study No. 9146V- 100355ARB, January 11, 2012. 

Unpublished. (MRID 48618001) 

Willoughby, J. (2012) MGK 264: estrogen receptor binding (rat uterine cytosol). CeeTox, Inc., 

Kalamazoo, MI. Laboratory Project Study No. 9146V- 100355ERB, January 11, 2012. 

Unpublished. (MRID 48618003) 

Willoughby, J. (2012) MGK 264: estrogen receptor transcriptional activation assay (human cell 

line (HeLa-9903)). CeeTox, Inc., Kalamazoo, MI. Laboratory Report No. 9146V-100355ERTA, 

January 11, 2012. Unpublished. (MRID 48618004) 

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Zorrilla, L. (2012) The hershberger bioassay for MGK 264 Technical. Integrated Laboratory 

Systems, Inc., Durham, NC. Laboratory Project Study No. C202-200, January 11, 2012. 

Unpublished. (MRID 48618005) 

Zorrilla, L. (2012) The uterotrophic assay for MGK 264 Technical. Integrated Laboratory Systems, 

Inc., Durham, NC. Laboratory Project Study No. C202-100, January 11, 2012. Unpublished. (MRID 

48618006) 

Zorrilla, L. (2012). Pubertal development and thyroid function with MGK 264 in 

juvenile/peripubertal female and male rats. Integrated Laboratory Systems, Inc., Durham, NC. 

Laboratory Study No. C202-300, April 12, 2012. Unpublished. (MRID 48686301). 

Weight of Evidence for MGK®-264: Assessment of Results of The Endocrine Disruptor Screen 

Program Tier I Battery. McLaughlin Gormley King Company 8810 Tenth Avenue North 

Minneapolis, MN 55427. MRID No. 49202101 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 48701701) of MGK 264 (95.4% purity) on amphibian metamorphosis 

of Xenopus laevis was conducted under flow-through conditions. Amphibian larvae at 

Nieuwkoop-Faber (NF) stage 51 (80 per control and treatment group) were exposed to a negative 

control and nominal concentrations of 0.011, 0.033 and 0.100 mg a.i./L. The time-weighted 

average (TWA) concentrations were <0.100 (<LOQ), 0.0171, 0.0390, and 0.106 mg a.i./L. The 

test system was maintained at 21.4 to 21.8oC and a pH of 7.3 to 7.9. 

The survival of tadpoles exposed to MGK 264 was not significantly affected (p>0.05) as survival 

was 100% in the control and low and mid treatment groups with one mortality occurring in the 

high treatment group. There were no clinical signs of toxicity or observations of abnormal 

behavior at any concentration. 

MGK 264 caused no significant acceleration or delay (p>0.05) of median NF developmental 

stage at Day 7 or 21 at any treatment level relative to the negative control. Further, no 

asynchronous development was observed. Nearly all individuals developed beyond NF stage 60 

by Day 21, with 95% (57/60), 87% (52/60), 82% (49/60), and 83% (49/59) NF≥61 individuals in 

the control, low, mid, and high treatment groups, respectively. The NF stage ranged from 61 to 

64 for late stage individuals, with a mode of NF stage 61 for all test levels, including the control. 

All other individuals (non-late-stage) were at NF stage 60 by Day 21. 

There was a significant increase in normalized (for snout-vent length) hind limb length (HLL) 

for Day 21 late-stage only individuals, at the high treatment level (Jonckheere-Terpstra, p<0.05), 

relative to the control. There were no other significant increases or decreases determined for Day 

7 normalized HLL or for Day 7 and Day 21 snout-vent length (SVL) and wet weight in late-stage 

tadpoles. A separate analysis conducted on those individuals not reaching stage 60 did not 

determine any significant effects; however the statistical power from this analysis is low due to 

the decreased number of tadpoles. 

Histopathological findings included mild follicular cell hypertrophy and hyperplasia in the 

negative control and all treatment groups. Specifically, for follicular cell hypertrophy, the 

number of mild diagnoses in the control group (7) was higher than the number for the mid and 

high treatment groups (6 and 5, respectively). Similarly for follicular cell hyperplasia diagnoses, 

there were more diagnoses of mild severity in the control group (5) than in the treatment groups 

(2, 1, and 0, respectively). 

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Although a majority of the surviving frogs were ≥NF stage 61 at test termination which may 

confound the ability to adequately evaluate potential thyroid-related effects in the AMA, there 

was no significant acceleration or delay of median NF developmental stage at any treatment 

concentration throughout the test nor effects on thyroid histopathology compared to the control. 

Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48618001), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment 

to measure the binding of a single concentration of [3H]-R1881 (1 nM) in the presence of 

increasing concentrations (10−10 to 10−3 M) of MGK 264 (95.4% a.i., Batch #AB1412). DMSO 

was used as the solvent at a final assay concentration of approximately 3.2%. A total of three 

independent runs were performed, and the assay included dexamethasone as a weak positive 

control, and R1881 as the ligand reference standard. 

The saturation binding experiment was conducted to demonstrate that the AR in the rat prostate 

cytosol was present in reasonable numbers and was functioning with appropriate affinity for the 

radio-labeled reference androgen (R1881) prior to conducting AR competitive binding 

experiments. Saturation binding data were not originally provided in the study report; however, 

summarized saturation binding data (MRID 48843501) from the performing laboratory were 

submitted following a request by the Agency. The mean dissociation constant (Kd) for [3H]-

R1881 was 0.613±0.041 nM and the estimated Bmax was 0.817±0.049 fmol/100 μg protein for 

the single batch of prostate cytosol that was prepared. The mean and individual Kd and Bmax

values were below the expected ranges reported in the EPA validation program (Kd: 0.685 to 

1.57 nM; and Bmax: 7 to 16 fmol/100 μg protein). 

 

In the competitive binding experiment, precipitation of MGK 264 was visually observed at the 

highest concentration (10−3

 M) tested in 2 of the 3 runs. In the first valid run, the mean specific 

binding was 48.7% at 10−3 M (without apparent precipitation), and 50.3% and 42.6% in the 2 

runs with precipitation noted at 10−3

 M. As data at 10−3 M were available from only one run, and 

were suspect because of precipitation at this concentration in the other two runs, the highest 

concentration used for data evaluation was 10−4

 M. The specific binding in the presence of 

MGK 264 was >75% at concentrations ≤10−4

 M in all three runs. An IC50 and Relative Binding 

Affinity (RBA) could not be calculated for MGK 264 as it did not result in 50% displacement at 

≤10−4 M. 

The estimated average log IC50 was −9.0 M for R1881 and −4.6 M for the weak positive control 

(dexamethasone). Compared to R1881, the mean RBA for dexamethasone was 0.004%. All 

performance criteria were met for two of the three runs. The Top % binding for MGK 264 and 

dexamethasone were above the recommended tolerance limit in the second run. However, as all 

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criteria for the weak positive control were met for the other two runs, it is unlikely that 

information from an additional run would change the interpretation of this study. 

Based on the results from the three runs, MGK 264 is classified as a Non-binder in the androgen 

receptor binding assay. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48618002), MGK 264 (95.4% a.i. Lot 

#AB1412) was incubated with human recombinant aromatase and tritiated androstenedione ([1β- 3

H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in 1% dimethyl sulfoxide (DMSO) at log 

concentrations from 10−11 to 10−3 M for 15 minutes to determine the potential for MGK 264 to 

inhibit aromatase activity. 

Aromatase activity was determined by measuring the amount of tritiated water produced at the 

end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified 

using liquid scintillation counting (LSC). For the three runs, each test concentration of MGK 

264 was tested in triplicate. Each run also included a full activity control, a background activity 

control, and a positive control series (10−10 to 10−5 M) using a known inhibitor 

(4-hydroxyandrostenedione; 4-OH ASDN). Because the highest concentration of MGK 264 

tested in Run 1 (10−3

 M) showed precipitation during the 15-minute incubation period, this 

concentration was not used for data interpretation. The highest successfully tested concentration 

for all three runs that was used for data interpretation was 10−4 M. 

Aromatase activity in the full activity controls was 0.611 ± 0.026 nmol·mg-protein−1

·min−1. The 

response of each full activity control within a run was between 90 to 110% of the average full 

activity. Activity in the background controls ranged from 0.25 to 0.37% and averaged 0.30% of 

the full activity control. 

For the positive control substance (4-OH ASDN), aromatase activity results were within the 

recommended ranges for the performance criteria. The estimated log IC50 for 4-OH ASDN 

averaged −7.27 M and the Hill slope was −0.94. 

For MGK 264, aromatase activity averaged 0.603 ± 0.026 nmol·mg-protein−1

·min−1 at the lowest 

tested concentration (10−11 M) and 0.062 ± 0.006 nmol·mg-protein−1

·min−1 at the highest 

successfully tested concentration (10−4

 M), corresponding to mean aromatase activity from 

97.76% to 10.22% relative to the full activity control. The estimated log IC50 for MGK 264 was 

−4.72 M and the estimated Hill slope was −1.0. 

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MGK-264 showed >50% inhibition of aromatase activity at the concentrations tested. Based on 

the data from the average response curve, MGK 264 is classified as an Inhibitor of aromatase 

activity in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48618003), uterine cytosol from Sprague 

Dawley rats was used as the source of ER to conduct binding experiments. The competitive 

binding experiment measured the binding of a single concentration of [3

H]-17β-estradiol (1 nM) 

in the presence of increasing concentrations (10−11 to 10−4

 M) of MGK 264 (95.4% a.i., Batch 

#AB1412). DMSO was used as the solvent at a final concentration of 4%. A total of three runs 

were performed, and each run included 19-norethindrone as a weak positive control, 

octyltriethoxysilane as a negative control, and 17β-estradiol as the natural ligand reference 

material. 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The protein concentrations used in the saturation binding 

runs varied between each run, and were approximately 3- to 6-fold greater than recommended 

(160 to 320 μg versus 50±10 μg). The Kd for [3H]-17β-estradiol was 0.331±0.061 nM and the 

estimated Bmax was 74.55±3.03 fmol/100 μg protein for the prepared rat uterine cytosol. The Kd

for each run was within the expected Guideline range of 0.03 to 1.5 nM. 

In the competitive binding experiment, the highest concentration tested in all three runs was 

10−4 M based on the preliminary precipitation assessment. The mean specific [3H]-17β-estradiol 

binding in the presence of MGK 264 was ≥85% at concentrations ≤10−4 M in all three runs. The 

estimated mean log IC50 and Relative Binding Affinity (RBA) were not calculated for MGK 264 

as the test chemical did not displace 50% of the radiolabeled ligand in any run. 

The estimated mean log IC50 was −9.0 M for the natural ligand (17β-estradiol) and −5.5 M for 

the weak positive control (19-norethindrone). Compared to the natural ligand, the mean RBA 

was 0.030% for 19-norethindrone. All performance criteria were met for 17β-estradiol and 19-

norethindrone; however, in two runs, octyltriethoxysilane displaced greater than 25% of [3

H]-

17β-estradiol from the ER at the highest concentration (10−3

M). 

Based on the results from the three runs, MGK 264 is classified as Not Interactive in the estrogen 

receptor binding assay. 

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ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48618004), hERα-HeLa-9903 

cells cultured in vitro were exposed to MGK 264 (95.4% a.i., Lot #AB1412) at logarithmically 

increasing concentrations from 10−11 to 10−4 M or 10−12 to 10−5 M in 0.1% DMSO for 

approximately 24 hours in two independent runs. The experiments were performed using 96-well 

plates and each MGK 264 concentration was tested in replicates of 6/plate in each run. Cells 

were exposed to the test agent for 24±2 hours at 37±2°C to induce reporter (luciferase) gene 

products. Luciferase expression in response to activation of the estrogen receptor was measured 

using a proprietary luciferase assay. 

MGK 264 was tested up to the limit of cytotoxicity, which was observed at 10−4

 M in the rangefinder and first run. The mean RPCMax for MGK 264 was 5.2% for the first run and 1.2% for the 

second run, and the associated PCMax was 10−7 M for the first run and 10−8 M for the second run. 

In the main assays, the responsiveness of the cells to the very weak positive control 17αmethyltestosterone was lower than the expected values, indicating a decreased sensitivity of the 

assay to very weak agonists. Although the conditions of this assay were not optimal to detect 

very weak activity, MGK 264 responses were similar to those of the negative control 

corticosterone (0.7-3.9% PC) and not comparable to the responses of 17α-methyltestosterone, 

which was able to reach a maximum of 8.1-52.8% PC. Cytotoxicity was observed at the highest 

dose of 17α-methyltestosterone tested (78.5% viability at 10−5 M). MGK 264 was only able to 

reach a maximum of 1.2-5.2% PC when tested up to the highest concentration possible based on 

cytotoxicity. The reference chemicals 17β-estradiol and 17α-estradiol (weak estrogen) 

responded as expected in both runs. Because the RPCMax < PC10 in both assay runs, MGK 264 

was considered negative for estrogen receptor transcriptional activation in this test system. 

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 48923701) of MGK 264 with fathead 

minnows (Pimephales promelas) was conducted under continuous flow-through conditions. 

Adult fish, 16 spawning groups (2 males and 4 females in each group; 4 groups/treatment; 4.5 - 6 

months old) were exposed to MGK 264 (95.4% purity) at nominal concentrations of 0 (control), 

0.011, 0.033, and 0.100 mg a.i./L; time-weighted average (TWA) concentrations were <LOQ 

(<0.001), 0.013, 0.036, and 0.107 mg a.i./L. The test system was maintained at 24.5 to 25.5o

C 

and a pH of 7.1 to 7.8. 

Adult male and female survival were unaffected by treatment with MGK 264; overall survival 

was 100% at all treatment levels. There were no notable observations with regards to behavior or 

changes in appearance, specifically color/banding, ovipositor appearance, or size of dorsal nape 

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pad in the control or treated groups or clinical signs of toxicity for any treatment group compared 

to the controls. Body weights and lengths in MGK 264-treated fish were similar to the control. 

Spawning occurred at least two times in 7 days and fecundity in the negative control was 32.4 

eggs/female/day/replicate (30.1 - 36.2); fertilization success in the negative control group 

averaged 99.1% (99.0 - 99.2%). Male gonado-somatic index (GSI) was the only endpoint to 

exhibit a treatment-related effect; there was a significant 45% increase in male GSI at the high 

treatment level, relative to the control (p<0.05; Dunnett’s test). There were no statistically 

significant differences (p>0.05) in treated groups compared to negative control for any other 

endpoint (i.e., female GSI, and male and female vitellogenin and nuptial tubercle score). 

Histopathology showed no treatment-related effects in males or females at any treatment level. 

However, it should be noted that female fish (including controls) had findings of oocyte atresia 

(5-14%) sometimes associated with inflammation (5-11%) and presence of protozoal spores. 

Male fish (including controls) had findings of increased proportion of spermatogonia (13-63%), 

and increases in testicular degeneration (38-63%) sometimes associated with inflammation 

(13%) or interstitial cell (Leydig) hypertrophy/hyperplasia (25%) or a testis-ova (13%) or duct 

mineralization (25%). There were no statistically significant differences between gonadal stages 

in treated groups compared to the negative controls. 

 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48618005) screening for androgenic activity, MGK 264 (95.4% 

purity, Batch/lot # AB1412) in corn oil was administered daily via oral gavage to groups of eight 

61/62-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 266, or 850 

mg/kg/day. An androgenic positive control group consisted of eight castrated rats exposed to the 

vehicle plus 0.4 mg/kg/day of testosterone propionate (TP) by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, MGK 264 in corn oil was administered daily via 

oral gavage to groups of eight 61/62-day old, castrated male Sprague Dawley rats at dose levels 

of 0 (vehicle), 85, 266, or 850 mg/kg/day in conjunction with a daily dose of reference androgen, 

TP, at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive control group consisted of 

eight castrated rats exposed to 3 mg/kg/day flutamide (FT, via oral gavage) and 0.4 mg/kg/day 

TP. The positive control group for the androgen agonist assay was used as the anti-androgenic 

negative control group. 

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For both components of the assay, body weights were determined daily. The animals were dosed 

for 10 consecutive days and terminated approximately 24 hours after the final dose 

administration. At necropsy, the five androgen-dependent tissues were collected and weighed. 

All animals survived until scheduled termination and no clinical signs of toxicity were observed 

in any animals 24 hours post dose. Body weights and overall (Days 1-11) body weight gains in 

the MGK 264 treatment groups were similar to controls in both the androgen agonist and antiandrogen assays. 

In the androgen agonist assay, body weights on Day 11 in the positive control (vehicle + TP) 

group were increased (p<0.05) by 8%, and overall body weight gains were increased (p<0.05) by 

63% compared to the vehicle controls. There were no treatment-related increases in any of the 

accessory sex organ weights of MGK 264-dosed animals. Animals in the positive control 

(vehicle + TP) group had accessory sex organ weight increases (p<0.05) as follows: 1120% in 

seminal vesicles; 1123% in ventral prostate; 220% in levator ani-bulbocavernosus muscle 

complex (LABC); 565% in Cowper’s glands; and 64% in glans penis. 

In the anti-androgen assay, body weights and overall body weight gains in the positive control 

(FT + TP) group were comparable to the negative controls. There were no treatment-related 

decreases in accessory sex organ weights for all treatment groups in the anti-androgen assay. 

Animals in the positive control (FT + TP) group had accessory sex organ weight decreases 

(p<0.05) as follows: 84% in seminal vesicles; 78% in ventral prostate; 55% in LABC; 60% in 

Cowper’s glands; and 25% in glans penis. 

The dosing for this study (high dose of 850 mg/kg/day) was considered adequate based on data 

from an earlier study showing clinical signs of toxicity and decreased body weights at 1000 

mg/kg/day. The performance criteria indicated that the assay performed as expected. 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissues. MGK 264 was negative for androgenicity and anti-androgenicity in the Hershberger 

assay. 

Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal assay (MRID 48686301), 16 Sprague-Dawley (Crl:CD®(SD) IGS) rats/dose 

group were treated daily via oral gavage (5 mL/kg) with MGK 264 (95.4% a.i., Lot #: AB1412) 

in corn oil at doses of 0, 400, or 800 mg/kg/day from post-natal day (PND) 22 to 42/43. Animals 

were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at day 

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of attainment were recorded. Following sacrifice on PND 42 or 43, blood was collected for 

clinical chemistry and hormone analyses. Total thyroxine (T4) and thyroid stimulating hormone 

(TSH) levels were determined using radioimmunoassays. Adrenals, liver, thyroid, pituitary, and 

urogenital organs were weighed and examined macroscopically. The uterus, left ovary, left 

kidney, and thyroid were examined microscopically. 

No adverse, treatment-related related effects were observed on survival, clinical signs, body 

weight, body weight gain, clinical chemistry, gross pathology, or uterine, ovarian, or renal 

histology. 

At 800 mg/kg/day, ages at initial and complete VO were delayed (p<0.001) by 3.1 and 3.4 days, 

respectively, and mean age of first estrus was delayed (p<0.05) by 2.5 days. Additionally, 

unadjusted and adjusted body weights at VO were increased (p<0.01) by 16-17%. VO occurred 

in all animals, and mean cycle length, % cycling, % regularly cycling, and the cycle status at 

necropsy were similar to controls at both doses. 

Serum T4 and TSH levels in the treated groups were similar to the controls. The following 

clinical chemistry parameters differed (p≤0.05) in the treated groups compared to controls: (i) 

potassium decreased by 8-11% at both doses; (ii) chloride increased by 2% each at both doses; 

(iii) calcium decreased by 4% each at both doses; (iv) creatinine decreased by 20% each at both 

doses; (v) SDH decreased by 37% at 400 mg/kg/day; (vi) ALP decreased by 28% at 800 

mg/kg/day; and (vii) total protein increased by 5% at 800 mg/kg/day. These differences were 

minor and all clinical chemistry values were within the reported normal range, with the 

exceptions of phosphorus, AST, ALT, ALP, and SDH. 

Significant decreases in mean colloid area and significant increases in mean follicular height 

were observed in the thyroid gland in animals administered 400 and 800 mg/kg/day MGK 264. 

There was an increased incidence of hyper-eosinophillic colloid (minimal to moderate severity) 

in the thyroids of females treated with 400 and 800 mg/kg/day MGK 264. However, serum T4 

and TSH levels were not affected, and there were no treatment-related effects on thyroid weight. 

Absolute, adjusted, and relative liver weights were increased (p<0.001) by 21-26% and 40-44% 

at 400 and 800 mg/kg/day, respectively. Additionally at 800 mg/kg/day, adjusted and relative 

pituitary weights were decreased (p<0.05) by 13 and 16%, respectively; absolute and adjusted 

ovary weights were decreased (p<0.05) by 13% each; and relative kidney weights were increased 

(p<0.05) by 5%. Absolute pituitary weight at 800 mg/kg/day was also decreased by 13%, but 

this decrease was not statistically significant. 

Although there were no adverse clinical signs or significant decreases in body weights observed 

at the high dose to indicate the high dose was at an adequate concentration, there was a positive 

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response (delayed VO and day of first estrus) observed at 800 mg/kg/day. Additionally, the 

findings observed at 1000 mg/kg/day in the range-finding and uterotrophic assays (mortality, 

abnormal behavior, lethargy, labored breathing, piloerection, hunched posture, and mild 

twitching) indicate that the high-dose of 800 mg/kg/day was appropriate. 

Male Pubertal Assay (OCSPP 890.1500) 

In a Male Pubertal assay (MRID 48686301), 16 male Sprague-Dawley (Crl:CD®(SD) IGS) 

rats/dose group were treated daily via oral gavage (5 mL/kg) with MGK 264 (95.4% purity, Lot 

#AB1412) in corn oil at doses of 0, 350, or 700 mg/kg/day from post-natal day (PND) 23 to 

53/54. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and 

age and body weight at day of attainment were recorded. Following sacrifice on PND 53 or 54, 

blood was collected for clinical chemistry and hormone analyses. Total serum testosterone, 

thyroxine (T4), and thyroid stimulating hormone (TSH) levels were determined using 

radioimmunoassays. Adrenals, liver, thyroid, pituitary, and urogenital organs were weighed and 

examined macroscopically. The thyroid, left kidney, left epididymides, and left testis were 

examined microscopically. 

No treatment-related related effects were observed on survival, clinical signs, body weights, final 

body weight, body weight gains, gross pathology, or testicular, epididymal, or renal histology. 

No treatment-related related effects were observed on body weight or age at PPS, age at partial 

PPS, proportion/incidence of un-separated preputia, androgen-dependent tissue organ weights, or 

serum testosterone levels. 

The following clinical chemistry parameters differed (p<0.05) in the treated groups compared to 

controls: (i) potassium decreased by 5-8% at both doses; (ii) ALP decreased by 17-19% at both 

doses; (iii) BUN increased by 17% at 700 mg/kg/day; (iv) SDH increased by 50% at 700 

mg/kg/day; and (v) total protein increased by 7% at 700 mg/kg/day. Creatinine increased by 

25% at both doses, but the increase was statistically significant only at 350 mg/kg/day. These 

differences were minor and, with the exception of ALP, were within the reported normal range. 

ALP levels at all doses, including controls, exceeded the reported normal range by 2.4-fold or 

more. The increase of ALP was slight, without clear dose-dependence, and was considered 

incidental. 

Absolute, adjusted, and relative liver weights were increased (p<0.001) by 20-23% and 39% at 

350 and 700 mg/kg/day, respectively. Gross pathology and clinical chemistry did not indicate 

hepatotoxicity. Additionally at 700 mg/kg/day, relative kidney weights were increased (p<0.01) 

by 8%, a minor effect. The adjusted kidney weights showed a linear trend of increased (p≤0.05) 

weight with increasing dose; however, no statistically significant effect was detected between the 

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treated groups and control using Dunnett’s test. Therefore, this linear trend was considered 

incidental. 

A significant decrease (p<0.05) in mean colloid area was observed at 350 mg/kg/day. The 

incidence/grade of colloid area at 700 mg/kg/day was similar to those observed at 350 

mg/kg/day, but the finding did not attain statistical significance. Follicular cell height was 

slightly increased at 350 and 700 mg/kg/day, but did not attain statistical significance. There 

was an increased incidence of hyper-eosinophillic colloid (minimal to moderate severity) in the 

thyroids of males treated with 350 and 700 mg/kg/day MGK 264. However, as serum T4 and 

TSH levels and thyroid weights were unaffected, the biological relevance of these histological 

changes is uncertain. 

Although the animals could have tolerated a higher dose, the high dose (700 mg/kg/day) was 

considered adequate, based on the information provided from the 14-day range finding study and 

the Hershberger assay. The investigators assumed that the decreased final body weights noted at 

1000 mg/kg/day after 14 days of administration (↓16%) and at 850 mg/kg/day after 10 days of 

administration (↓5%) may have been more severe after 31 days of administration in the male 

pubertal assay. Additionally, 700 mg/kg/day approached the limit dose of 1000 mg/kg/day, 

considering the lack of a steep dose-response curve. No clearly adverse effect was noted in this 

study, but a treatment-related (possibly adaptive response) was observed on liver weight. 

Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48672301), H295R cells cultured in vitro in 24-well plates 

were incubated with MGK 264 (95.4% purity, Lot # AB1412) at log concentrations from 0.0001 

to 100 μM for 48 hours in triplicate for four independent experiments. Dimethyl sulfoxide 

(DMSO) was used as the vehicle, at a final concentration in the assay of 0.05%. Because 

precipitation and cytotoxicity was observed at 100 μM of MGK 264, this concentration was not 

evaluated statistically. In addition, Run 1 was not analyzed because one of the forskolin 

concentrations on the quality control plate was incorrect (3.3 μM was used instead of 1 μM). 

Testosterone and estradiol levels were measured using HPLC-MS/MS with internal standards. A 

Quality Control (QC) plate was run concurrently with each independent run of a test chemical 

plate to demonstrate that the assay responded properly to positive control agents at two 

concentration levels; positive controls included the known inhibitor (prochloraz) and inducer 

(forskolin) of estradiol and testosterone production. 

Guideline acceptability recommendations and requirements were generally met, including 

adequate production of testosterone and estradiol, acceptable reproducibility (low %CV), and 

appropriate induction and inhibition with positive controls. 

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Statistically significant inhibition of testosterone was noted in Run 4 at 10 μM of MGK 264, and 

induction of estradiol was observed in Run 3 at 0.1 and 1 μM. However, these effects were not 

reproducible in this assay in the other runs, and the response was similar to the solvent control 

(SC) when normalized for cell viability. Additionally, the apparent effect on estradiol was not 

concentration-dependent. Based on these findings, MGK 264 did not inhibit or induce hormone 

production under the conditions for this assay. 

Based on hormone responses in at least three independent runs, MGK 264 treatment did not 

result in statistically significant and reproducible alterations in testosterone or estradiol 

production 

Uterotrophic Assay (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48618006) conducted to screen for potential estrogenic activity, 

MGK 264 (95.4% purity, Batch/lot # AB1412) in corn oil was administered daily via oral gavage 

to groups of eight ovariectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 320, 

or 1000 mg/kg/day on post-natal days (PND) 56-58. A positive control group was treated with a 

daily dose of 17α-ethynyl estradiol (EE) in corn oil at 0.05 mg/kg/day by oral gavage. Body 

weights were determined daily. All animals were terminated and necropsied on PND 59 

approximately 24 hours after the final dose administration to determine wet and blotted uterine 

weights. 

One animal from the 1000 mg/kg/day group was euthanized moribund on Day 3; all other 

animals survived until scheduled termination. Animals in the high dose MGK 264 group 

exhibited adverse clinical signs of toxicity (uncoordinated movement, decreased movement, 

hunched posture, twitching, piloerection, ungroomed appearance, a red nasal discharge, and/or 

diarrhea) following dosing. 

Body weights were decreased (p<0.05) by 10% on Day 4 in the 1000 mg/kg/day group compared 

to the vehicle controls, resulting in a mean overall body weight loss (p<0.05) of -8.9 g, compared 

to the vehicle control group (16.5 g gain). Final body weights and overall body weight gains in 

the 320 mg/kg/day group were comparable to controls throughout the duration of the assay. 

Uterine weights in the MGK 264 treated groups were comparable to the vehicle controls. 

The body weights and overall body weight gains for the positive control (EE) group were not 

statistically evaluated; however, there was a mean overall body weight loss of −2.4 g over the 

duration of the study. Absolute wet and blotted uterus weights for the positive control (EE) 

group were each increased (p<0.05) by 99%, as expected. 

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Dosing for the assay was adequate, as animals were dosed up to the limit dose (1000 mg/kg/day), 

which resulted in decreased body weight and adverse clinical signs. 

No statistically significant changes were seen in uterine weight in this assay. MGK 264 is 

negative in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 general toxicity studies considered relevant for determining the 

potential interaction of MGK 264 with the endocrine system are summarized below. Given the 

breadth of toxicity data available on MGK 264, the only Part 158 studies discussed in this 

appendix are those that fulfill current guideline requirements and are directly relevant to 

assessing potential endocrine effects (i.e., multiple-generation rodent reproductive studies, 

development studies, chronic toxicity studies, etc.). 

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation reproduction toxicity study (MRID 48084901), groups of 25 Wistar rats/sex 

were administered MGK 264 Insecticide Synergist (94.2% purity) in the diet at dietary levels of 

0, 400, 1600, or 6400 ppm (equivalent to 0/0, 36/52, 159/225, and 650/952 mg/kg/day in 

males/females) for two consecutive generations. The P generation animals were fed the diets 

beginning 10 weeks prior to mating to produce the F1 litters. F1 offspring (25/sex/dose group) 

selected to be parents of the next generation received the same doses as their parents. One set of 

litters (F1) was produced by the P parents, and the F1 adults produced two litters (F2a and F2b). 

At 6400 ppm, F1 male and female body weights were decreased (p<0.05) by 6% at Week 17, and 

Week 5-17 body weight gains were decreased (p<0.05) by 5%. Relative (to body weight) food 

consumption was increased (p<0.05) by 11% during Weeks 5-16. In the 1600 ppm F1 females 

during the 2nd mating, body weights were decreased (p<0.05) by 5% at the end of gestation. At 

6400 ppm, body weights were decreased (p<0.05) by 8-10% at the end of both gestations and on 

post-natal day (PND) 4. Relative food consumption was dose-dependently increased in the 1600 

and 6400 ppm F1 dams during the 2nd mating. 

There were no effects of treatment on uterus, testes, epididymides, pituitary, or thyroid weights 

or microscopic pathology. Absolute and relative liver weights were dose-dependently increased 

in both generations in both sexes. With the exception of the 1600 ppm F1 females, these 

increases were statistically significant (p<0.05) in both sexes from both generations at 1600 and 

6400 ppm. At 400 ppm, the only increases were in the P and F1 males. Liver enlargement was 

found in 10 P females and 12 F1 females at 6400 ppm compared to 0 controls. In the P rats, 

increased incidences of distinct lobulation of the liver were observed at 1600 ppm (9 males, 4 

females) and 6400 ppm (10 males, 8 females) compared to 0 controls. Incidences of centrilobular 

hypertrophy and eosinophilic centrilobular cytoplasmic changes in the liver were dosedependently increased at 1600 and 6400 ppm in both sexes from both generations. Centrilobular 

hypertrophy was found at these doses in the males (16-25 treated) and females (9-24 treated), 

and eosinophilic centrilobular cytoplasmic changes were noted in the males (9-25) and females 

(9-24), all compared to 0 controls. Additionally at 6400 ppm, periportal hypertrophy was noted 

in 21 P females. Deposition of brownish pigment in/around bile ducts with prominent bile duct 

epithelium, and periductular cellular infiltration and/or degeneration of bile duct epithelium were 

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reported at 6400 ppm in both sexes of both generations. Presence of hydropic foci was noted in 

both generations of males at 6400 ppm. There was an increased amount of 

periportal/centrilobular fat storage in both generations of males at 1600 ppm and above and in 

females at 6400 ppm. 

In the kidney, increased storage of brownish pigment in the cortical tubules was observed at 

1600 ppm and above in both sexes and generations. Increased severity in basophilic cortical 

tubules hyaline droplets were noted in P males at 6400 ppm. In the thyroid glands, increased 

incidences of follicular cell hypertrophy were observed in both generations in the 1600 ppm 

males and 6400 ppm females. Increased incidences of colloidal alteration were noted in the P 

males at 6400 ppm, in the F1 males at 1600 and 6400 ppm, and in the F1 females at 6400 ppm. 

Altered luteinization of the corpora lutea was observed at 6400 ppm in the P (8/25) and F1

(11/25) females compared to 0/25 controls. There were an increased number of F1 females in 

met-/diestrus cycle at 6400 ppm (16/25 treated vs 8/25 controls). Additionally at 6400 ppm in the 

P generation: absolute and relative ovary/oviduct weights were significantly (p<0.01) decreased 

by 16-20%, and absolute and relative seminal vesicle/coagulating gland weights were increased 

by 9-10% (p<0.05). However, because there were no corroborating microscopic findings and no 

effect was found on sperm parameters, insemination, or fertility, the increased seminal 

vesicle/coagulating gland weights were not considered adverse. 

The parental LOAEL is 1600 ppm (equivalent to 159/225 mg/kg/day) based on decreased body 

weights in females and adverse effects on the liver, kidneys, and thyroid in both sexes. The 

NOAEL is 400 ppm (equivalent to 36/52 mg/kg/day in males/females). 

Live birth index, sex ratio, number of pups born, prenatal losses, and viability and lactation 

indices were unaffected by treatment. 

In the F2a litters, small pups relative to other pups in the litter were observed with a higher 

frequency at 6400 ppm compared to controls. At 6400 ppm in both sexes of the F2b litters, a nonstatistically significant reduction in body weight was observed for PND 1-7. Except for the F2a

males on PND 21, significantly (p≤0.05) decreased offspring body weights were observed in 

both sexes in the F1, F2a, and F2b litters on PND 21 and 28. Additionally, in the F2b females, body 

weights were decreased by 14% (p<0.05) on PND 14. 

Body weight at the time of balano-preputial separation was significantly lower (p<0.01) at 6400 

ppm; however, the time to sexual maturation was unaffected by treatment, indicating that the 

decreased growth did not affect sexual maturation. Furthermore, the mean body weight of the 

6400 ppm males at the time of sexual maturation was within the historical control range. In 

contrast, vaginal opening was delayed (p<0.01) by 4.2 days in the F1 generation. Although not 

statistically significant, the body weights at vaginal opening were dose-dependently increased, 

indicating that sexual maturation in the females was not secondary to growth (body weight). 

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Therefore, a direct effect on sexual maturation cannot be discounted. There was no effect on 

anogenital distance in either males or females in the study. 

The offspring LOAEL is 6400 ppm (equivalent to 650/952 in males/females) based on decreased 

pup body weights and delayed vaginal opening. The NOAEL is 1600 ppm (equivalent to 

159/225 mg/kg/day in males/females). 

There were no treatment-related effects on estrus cycle, sperm parameters, insemination, fertility, 

pre-coital interval, gestation, rearing, gestation duration, or number of litters born. At 6400 ppm, 

there was a small but significant decrease in implantations with concomitant lower mean litter 

size at birth in F1 pups. Although the mean litter size is within the historical control range, a 

relationship to treatment cannot be ruled out due to the histopathological findings (altered 

luteinization) in the parental females. 

The reproductive LOAEL is 6400 ppm (equivalent to 650/952 in males/females) based on 

decreased number of implantations and litter size, possibly related to effects on the 

ovaries/corpora lutea. The NOAEL is 1600 ppm (equivalent to 159/225 mg/kg/day in 

males/females). 

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

In a multi-generation reproduction toxicity study (MRID 42155701), groups of 26 SpragueDawley rats/sex were administered MGK 264 (100% purity) in the diet at dose levels of 0, 2500, 

10,000 or 20,000 ppm. However, high mortality was observed in the 20,000 ppm group, so this 

group was terminated, and a second control group and a 1250 ppm group were initiated 

approximately two months after study initiation. These dose levels (0, 1250, 2500 and 10,000 

ppm) correspond to mean doses of approximately 0/0, 61/73, 125/151, and 649/759 mg/kg/day in 

males/females. The diets were administered to the P generation for at least 80 days prior to 

mating for the F1a litters, and to the F1 parents for at least 102 days prior to mating for the F2a

litters. 

There were no treatment-related mortalities or clinical signs. Organ weights were not reported. 

There were no effects of treatment reported for microscopic findings in the ovaries, testes, or 

epididymides. 

Body weight decreases (p<0.05) during pre-mating were noted at 1250 (↓5-10%), 2500 (↓5-7%), 

and 10,000 (↓9-15%) ppm in the F1 dams and at 2500 (↓↓6-8%) and 10,000 (↓6-10%) ppm in the 

P dams. In the F1 males, body weighs were decreased (occasionally p<0.05) at 2500 (↓4-8%) and 

10,000 (↓9-20%) ppm. Hepatocellular hypertrophy was evident at all doses in the F1 parental 

group and at higher levels in the P parental group. The parental LOAEL is 1250 ppm (equivalent 

to 61/73 mg/kg/day in males/females), based on decreased body weight and hepatocellular 

hypertrophy. The parental NOAEL was not established. 

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Decreased body weight during lactation was evident at all doses with there being decreases of 

about 5% to 14% (all p < 0.05) at 1,250 ppm, 5% to 13% at 2,500 ppm and 26% to 31% at 

10,000 ppm. Following lactation, the pups gained weight comparable to the controls. Because of 

a variety of factors including lack of a true dose response between 1250 and 2500 ppm, the fact 

that the pups are taking in large quantities of milk and feed and because the difference was 

relatively small and reversible, the dose level of 1250 ppm is considered to be near the NOAEL 

and additional testing is not considered necessary. The developmental LOAEL is 2500 ppm 

(equivalent to 61/73 mg/kg/day in males/females), based on slight decreases in body weight. The 

developmental NOAEL was not established. 

There were no adverse effects on reproductive performance. The reproductive performance 

LOAEL was not observed. The reproductive NOAEL is 10,000 ppm (equivalent to 649/759 

mg/kg/day in males/females). 

Developmental Toxicity (Rat, OCSPP 870.3700) 

In a developmental toxicity study (MRID 41660302), groups of 25 presumed pregnant 

COBS®CD® rats were administered MGK 264 (98% purity) in 0.5% methylcellulose (dose 

volume 10 mL/kg) by daily oral gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day during 

gestation days (GD) 6-15. Dams were euthanized on GD 20 by carbon dioxide inhalation, and 

the fetuses removed by cesarean section. Fetuses were weighed, sexed, and examined for 

external, visceral, and skeletal alterations. 

There was one death in the 100 mg/kg/day group and one death in the 1000 mg/kg/day group; all 

other rats survived to scheduled euthanasia. Increased salivation was noted at 1000 mg/kg/day as 

a pre-dose observation in four dams and post-dosing in 23 dams. Body weight gains were 

decreased by 25% (p<0.01) during GD 6-15 at 1000 mg/kg/day. The maternal LOAEL was 1000 

mg/kg/day, based decreased body weight gain. The maternal NOAEL is 300 mg/kg/day. 

The pregnancy rate was decreased at 1000 mg/kg/day (68% treated vs. 80% controls); however, 

there was no corroborating evidence of effects on fertility. There were no treatment-related 

external, visceral, or skeletal alterations observed. The developmental LOAEL was not observed. 

The developmental NOAEL is 1000 mg/kg/day. 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a developmental toxicity study (MRID 40352301), groups of 16 New Zealand white rabbits 

were administered MGK 264 (98% purity) in 0.5% methylcellulose by daily oral gavage at dose 

levels of 0, 10, 30, or 100 mg/kg/day during GD 7-19. Does were euthanized on GD 29, and the 

fetuses removed by cesarean section. Fetuses were weighed, sexed, and examined for external, 

visceral, and skeletal alterations. 

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There were no treatment-related effects on mortality, body weight gains, parental gross necropsy 

or cesarean parameters. One doe died on study in both the 10 and 30 mg/kg/day groups. There 

was one abortion each in the control, 10, and 100 mg/kg/day groups. There were three abortions 

in the 30 mg/kg/day group including an abortion in the animals that died (this animal was said to 

have aborted and then died). An increase in the incidence of excessive salivation was seen at 100 

mg/kg/day (44% of the does vs. 0% in the controls); this occurred on consecutive days in the 

middle of the dosing period and is considered a response to treatment. The maternal LOAEL for 

maternal toxicity is 100 mg/kg/day based on excessive salivation. The maternal NOAEL is 30 

mg/kg/day. 

There were no effects of treatment on fertility or on external, visceral, or skeletal alterations. The 

developmental LOAEL was not observed. The developmental NOAEL was 100 mg/kg/day. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a chronic toxicity/carcinogenicity study (MRID 43005301), groups of 60 CD rats/sex were 

administered MGK 264 (100% purity) in the diet at doses of 0 (two separate groups), 50, 150, or 

450 mg/kg/day for 104 weeks. 

There were no effects of treatment on mortality, clinical signs, ophthalmoscopic examinations, 

hematology, clinical chemistry, or urinalysis. No treatment-related changes were seen in absolute 

or relative weights of the testes, ovaries or adrenal glands, and no treatment-related 

histopathological lesions were seen in the testes, epididymides, or ovaries. 

At 450 mg/kg/day, body weights were decreased (p<0.05) after Week 18 in males and Week 10 

in females, with final body weights decreased 8-9% in males and 18-21% in females, compared 

to the two control groups. Decreases in body weight gains were also observed at this dose, 

reaching 58% in males and 50% in females during Weeks 52-78. Food consumption was also 

generally decreased in females at 450 mg/kg/day. 

Both absolute and relative liver weights were dose-dependently increased in males and females. 

Compared to the two control groups, both absolute and relative liver weights were increased in 

females at 150 mg/kg/day by 9-20% (NS) and 22-24% (NS), respectively, and at 450 mg/kg/day 

by 22-34% (p<0.05) and 61-63% (p<0.01), respectively. In males, absolute and relative liver 

weights were also increased at 50 mg/kg/day by 9-14% (p<0.05) and 9-12% (NS), respectively, 

at 150 mg/kg/day by 10-16% (p<0.05) and 18-21% (p<0.05), respectively, and at 450 mg/kg/day 

by 39-45% (p<0.01) and 55-59% (p<0.01), respectively. At dose levels ≥150 mg/kg/day, 

pathological changes were observed in the liver (hepatocyte hypertrophy in both sexes and portal 

bile duct proliferation and bile stasis, spongiosis hepatitis and cysts in females) and kidney 

(brown pigment in females). At 450 mg/kg, additional pathology was noted in the liver (bile duct 

proliferation and cysts in both sexes, and altered eosinophilic foci and spongiosis hepatitis in 

males) and kidney (brown pigment and cysts in males); and kidney, heart, and brain weights 

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were increased. The systemic LOAEL is 150 mg/kg/day based on increased liver weight 

supported by liver pathology (hepatocyte hypertrophy and other liver histopathology). The 

NOAEL is 50 mg/kg/day. 

An increase in thyroid follicular tumors in males was noted. A response of 2 (3.3%), 1 (1.7%), 4 

(6.7%), 5 (8.3%) and 6 (10%) for adenomas alone, and 4 (6.7%), 1 (1.7%), 6 (10%), 7 (11.7%) 

and 9 (15%) for combined adenomas and carcinomas for the two control, 50, 150 and 450 

mg/kg/day dose groups respectively (N=60/dose group). 

Carcinogenicity (Mouse, OCSPP 870.4200) 

In a carcinogenicity study (MRID 42093802), groups of 50 CD-1 mice/sex were administered 

MGK 264 (98% purity) in the diet at 0 (two separate groups), 50, 400, or 800 mg/kg/day for 18 

months. 

There were no treatment-related effects observed on mortality, clinical signs of toxicity, body 

weights, food consumption, ophthalmological parameters, or hematology parameters at any dose 

in either sex. 

No treatment-related changes were seen in the absolute or relative (to body) weights of the testes, 

ovaries, or adrenal glands. At 400 mg/kg/day and above, absolute and relative (to body) liver 

weights were increased (p<0.01) in the males by 13-37% and 22-46%, respectively, compared to 

the controls. In the females, only relative (to body) liver weight at these doses was significantly 

(p<0.01) increased (↑11-16%), although absolute liver weights were slightly elevated at 400 

(↑8%) and 800 mg/kg/day (↑6%). 

No treatment-related histopathological findings were seen in the testes, epididymides, seminal 

vesicle, prostate, ovaries, uterus, mammary, thyroid, adrenal or pituitary glands. 

At 400 mg/kg/day there was increased calculi in the gall bladder and intrahepatic bile stasis in 

males. At 800 mg/kg/day there was an increase in hepatocellular hypertrophy, calculus in bile 

duct in males and females and portal bile duct proliferation, portal mononuclear cell infiltration, 

spongiosis hepatis, cysts and vacuolar change in males and hyperplastic nodules in males and 

females. 

The 400 and 800 mg/kg/day doses were associated with increases in liver adenomas in males. In 

females, the 400 and 800 mg/kg/day doses had adenomas and a carcinoma which did not reach 

statistical significance, but these liver tumors are rare in female CD-1 mice. 

The systemic LOAEL was 400 mg/kg/day based on increased absolute and relative liver weight 

and liver pathology findings in males. The systemic NOAEL was 50 mg/kg/day. 

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Chronic Toxicity (Dog, OCSPP 870.4100) 

In a one-year chronic toxicity study (MRID 42148102), groups of 4 beagle dogs/sex were 

administered MGK 264 (92% purity) in the diet at concentrations of 0, 65, 250, or 1000 ppm 

(equivalent to 0/0, 2.1/2.0, 7.5/7.4, and 34.9/32.5 mg/kg/day in males/females) for one year. 

There were no effects of treatment on mortality, clinical signs, body weight gains, food 

consumption, ophthalmoscopic examinations, hematology, clinical chemistry, urinalysis, or 

gross pathology. There were no treatment-related effects on organ weights or microscopic 

pathology in the ovary, testes, pituitary, adrenal, or thyroid. 

The only effects noted were seen at the 1000 ppm dose level and consisted of brown pigment 

deposition in the liver of both sexes (3/4 males and females vs. 0/4 in controls or lower doses) 

and hepatocellular hypertrophy in 2/4 males. The LOAEL is 1000 ppm (equivalent to 34.9/32.5 

mg/kg/day in males/females), based on liver effects. The NOAEL is 250 ppm (equivalent to 

7.5/7.4 mg/kg/day in males/females). 

Subchronic Oral Toxicity (Rat, OCSPP 870.3100) 

In a subchronic toxicity study (MRID 42970601), groups of 10 Sprague-Dawley rats/sex were 

administered MGK 264 (98% purity) in the diet at nominal dose levels of 0, 125, 250, 500, 1000, 

or 2000 mg/kg/day for 13 weeks. Actual achieved doses were 0, 140, 279, 564, 1124 and 1842 

mg/kg/day in males and 0, 130, 260, 522, 1034 and 1933 mg/kg/day in females. 

Clinical signs of toxicity were observed in high-dose male and females, including decreased 

defecation in all animals, discolored urine in all males and 8 females, anogenital staining in 4 

males and one females, and labored breathing in 2 males and 6 females. In addition, high levels 

of mortality were observed at the high dose, with three males and six females dying on study or 

being sacrificed in extremis. Because of this excessive mortality, the surviving animals in this 

group were removed from test-article containing diet and placed on basal diet prior to the end of 

the study. Mortality in this group was likely due to decreased food consumption (50% decrease 

in food consumption in males and 35% in females at week one) resulting from an unpalatable 

diet. Once removed from the test diet the surviving animals in this group rapidly gained weight 

and appeared healthy. There were no premature deaths or clinical signs in either sex of any other 

test group. 

There were no effects of treatment on hematology, testes, ovary, or adrenal weights, or on gross 

pathology of the testes, epididymides, ovary, or adrenal. None of the collected tissues were 

examined for histologically. 

Terminal body weights (Week 13) were decreased by 7 and 9% (NS) in males and females at 

500 mg/kg/day, respectively, and by 14 and 17% (p<0.05) in males and females at 1000 

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mg/kg/day. Body weight gains were also decreased at 500 and 1000 mg/kg/day in both sexes 

compared to controls (↓10% and ↓21% in males, and ↓18% and ↓33% in females). Food 

consumption was frequently decreased (p<0.05) at 500 and 1000 mg/kg/day in males and 

females throughout the study. 

In males, dose-dependent increases were observed in mean total protein values (↑7-11%) and 

globulin levels (↑14-24%) at 250 mg/kg/day and above, and in cholesterol levels (↑22-67%) at 

500 mg/kg/day and above. In females, dose-dependent increases were also observed in mean 

total protein values (↑8-18%), globulin levels (↑25-38%), and cholesterol levels (↑26-89%) at 

500 mg/kg/day and above. 

Dose-dependent increases were observed in absolute and relative (to body) liver weights in 

males and females at doses from 125 to 1000 mg/kg/day, with the changes becoming significant 

(p<0.05) at ≥250 mg/kg/day. Absolute liver weights were increased (p<0.05) by 6% (NS) to 

56% in males and by 8% (NS) to 61% in females, and relative (to body) liver weights were 

increased (p<0.05) by 8 to 104% in males and by 6 (NS) to 84% in females. 

The only findings observed at gross necropsy that were likely related to compound exposure 

were findings of enlarged liver in males (0% in controls compared to 30% at 500 and 20% at 

1000 mg/kg/day) . 

The NOAEL is 125 mg/kg/day. The LOAEL is 250 mg/kg/day based on evidence of liver 

alterations (increased serum globulins and total proteins in males, and increased absolute and 

relative liver weights in both sexes). 

Subchronic Inhalation Toxicity (Rat; OCSPP 870.3465)

In a subchronic inhalation toxicity in rats (MRID 43309001), groups of 15 Sprague-Dawley 

rats/sex were exposed to atmospheres containing MGK 264 at concentrations of 0, 0.010, 0.040, 

0.135 or 0.400 mg/L for three months (5 days/week, 6 hours/day). An additional 15 rats/sex were 

exposed to MGK 264 at concentrations of 0 or 0.400 mg/L as above and maintained unexposed 

for three additional months in a recovery phase. 

There were no effects of treatment on body weights, body weight gains, food consumption, 

ophthalmoscopic examinations, hematology, clinical chemistry, absolute or relative (to body) 

organ weights, or gross or microscopic pathology with the exceptions of the nasoturbinates, 

nasopharynx, and larynx. The ovaries, testes, and epididymides weights and testes and 

epididymides pathology were unaffected by treatment. 

There were no treatment-related deaths. One control male died on Day 46; a probable cause of 

death was chronic pyelitis affecting the kidneys. One high-dose male died during the recovery 

period; a cause of death was not determined. Clinical signs of toxicity observed were red facial 

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stains in all treated females and in males at ≥0.040 mg/L, and decreased activity in the 0.400 

mg/L group. 

In the larynx, metaplasia/hyperplasia and metaplastic keratinized epithelium of the mucosa were 

present in all dosed groups, and the severity of these lesions increased with the dose level. 

Stratified squamous epithelium (normal) hyperplasia and hyperkeratosis were present in the midhigh and high dose groups. Ventral diverticulum: pseudostratified cuboidal/columnar epitheliumsquamous/squamoid metaplasia/hyperplasia was also reported to be present only in the mid-high 

dose (3 males and 4 females) and high-dose (6/sex) groups. Mucosal metaplasia/hyperplasia was 

minimally present in 12 males and 8 females in the high-dose group after the recovery period. 

The keratinized tissue was still present in two high dose males only (minimal degree) also 

implying a slow rate of recovery. The normal hyperplasia and hyperkeratosis were also present 

to minimum degree in one or two of the high dose group further indicating a slow recovery. 

In the nasoturbinates, the mid-high and high dose groups were associated with increases in 

frequency (for females) and intensity (for both sexes) of eosinophilic material in the epithelium. 

The severity of hypertrophy/hyperplasia of goblet cells in the respiratory epithelium was 

increased in the high dose group. The hypertrophy/hyperplasia incidence and severity scores for 

the control and 0.400 mg/L groups suggested that the condition is reversed in males but persisted 

in females. The eosinophilic material incidence and severity scores indicated reversal after the 3 

month recovery period. 

In the nasopharynx hypertrophy/hyperplasia of the goblet cells in the epithelium was increased in 

frequency in the 0.400 mg/L group. Recovery was observed. 

Subchronic Dermal Toxicity (Rabbit, OCSPP 870.3250) 

In a subchronic dermal toxicity study (MRID 42742201), groups of 10 New Zealand white 

rabbits/sex were administered MGK 264 (92% purity) in corn oil (1 mL/kg) by dermal 

application to the shaved, intact skin at concentrations of 0, 10, 30, or 100 mg/kg for 6 hours/day, 

7 days/week for 13 weeks. No treatment-related effects were observed on mortality, clinical 

signs, body weights, food consumption, ophthalmoscopic parameters, hematology and clinical 

chemistry parameters, organ weights (liver, testes, or kidneys), or gross and histopathological 

findings (including testes, epididymides, and ovaries). Signs of epidermal hyperplasia, 

hyperkeratosis, scab formation, chronic inflammation, and hemorrhage were noted in all groups 

without evidence of compound-related effects, and were considered to be related to the corn oil 

vehicle and/or bandaging. 

The systemic LOAEL was not observed. The systemic NOAEL was 100 mg/kg/day. 

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